Method for designing protein kinase inhibitors

ABSTRACT

The present invention provides a method for identifying inhibitors of protein kinases. Methods are also provided for inhibiting protein kinase activity. Specific non-peptide protein tyrosine kinase inhibitor are provided. The protein kinases produced using the method of the present invention may be used to treat a number of conditions in patients, including cancer, psoriasis, arthrosclerosis, or immune system activity.

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/115,643, filed Jan. 13, 1999.

BACKGROUND OF THE INVENTION

Protein kinases are a large class of enzymes which catalyze the transferof the γ-phosphate from ATP to the hydroxyl group on the side chain ofSer/Thr or Tyr in proteins and peptides and are intimately involved inthe control of various important cell functions, perhaps most notably:signal transduction, differentiation and proliferation. There areestimated to be about 2,000 distinct protein kinases in the human body(Hunter, 1987, 1994, Hanks & Hunter, 1995), and although each of thesephosphorylate particular protein/peptide substrates, they all bind thesame second substrate ATP in a highly conserved pocket.

Inhibitors of various known protein kinases could have a variety oftherapeutic applications provided sufficient selectivity, and acceptablein vivo pharmacological properties, can be incorporated into suchinhibitors (Levitzki, 1996a). Perhaps the most promising potentialtherapeutic use for protein kinase inhibitors is as anti-cancer agents.This potential application for protein tyrosine kinase (“PTK”)inhibitors has been highlighted in many recent reviews (e.g. Lawrence &Hiu, 1998, Kolibaba & Druker, 1997, Showalter & Kraker, 1997, Patrick &Heimbrook, 1996, Groundwater et al., 1996, Levitzki, 1995). Thefoundation for this application is based partly upon the fact that about50% of the known oncogene products are PTKs and their kinase activityhas been shown to lead to cell transformation (Yamamoto, 1993).

The PTKs can be classified into two categories (Courtneidge, 1994), themembrane receptor PTKs (e.g. growth factor receptor PTKs) and thenon-receptor PTKs (e.g. the src family of proto-oncogene products).There are at least 9 members of the src family of non-receptor PTK'swith pp60^(c-src) (hereafter referred to simply as “src”) being theprototype PTK of the family wherein the ca. 300 amino acid catalyticdomains are highly conserved (Rudd et al., 1993, Courtneidge, 1994). Thehyperactivation of src has been reported in and number of human cancers,including those of the colon (Mao et al., 1997, Talamonti et al., 1993),breast (Luttrell et al., 1994), lung (Mazurenko et a, 1992), bladder(Fanning et al., 1992) and skin (Barnekow et al., 1987) as well as ingastric cancer (Takeshima et al., 1991), hairy cell leukemia (Lynch etal., 1993) and neuroblastoma (Bjelfman et al., 1990). Overstimulatedcell proliferation signals from transmembrane receptors (e.g. EGFR andp185HER2/Neu) to the cell interior also appears to pass through src (Maoet al., 1997, Parsons & Parsons, 1997, Bjorge et al., 1996, Taylor &Shalloway, 1996). Consequently, it has recently been proposed that srcis a universal target for cancer therapy (Levitzki, 1996) because its'hyperactivation (without mutation) is involved in tumor initiation,progression and metastasis for many important human tumor types.

In view of the large, and growing, potential for inhibitors of variousprotein kinases, a variety of approaches to obtaining useful inhibitorsis needed. The status of the discovery of PTK inhibitors (Lawrence &Niu, 1988, Showalter & Kraker, 1997, Patrick & Heimbrook, 1996,Groundwater et al., 1996, Budde et al., 1995, Levitzki & Gazit, 1995)has been extensively reviewed. Random screening efforts have beensuccessful in identifying non-peptide protein kinase inhibitors but thevast majority of these bind in the highly conserved ATP binding site. Anotable recent example of such non-peptide, ATP-competitive, inhibitorsare the 4-anilinoquinazolines, and analogs, which were shown to beeffective against the epidermal growth factor receptor PTK (EGFRTK)(e.g. Rewcastle et al., 1996). Although this class of inhibitors wasreported to be selective for the EGFR PTK vs. six other PTKs (includingsrc, Fry et al., 1994) it is unknown what their effect is on most of theremaining 2,000 protein kinases that all bind ATP as well as a largenumber of other ATP, ADP, GTP, GDP, etc. utilizing proteins in the body.Therefore, potential side effects from PTK inhibitor drugs that mimicATP, which might only be discovered after expensive animal toxicitystudies or human clinical trials, are still a serious concern. Also,although this class of compounds was a nice discovery and is undergoingfurther exploration, they do not provide a rational and general solutionto obtaining non-peptide inhibitors for any desired PTK, e.g. in thiscase src. The risk of insufficient specificity in vivo withATP-competitive PTK inhibitors has also been noted by others, along withthe inherent three order of magnitude reduction in potency theseinhibitors display when competing with the mM levels of intracellularATP rather than the μM levels used in the isolated enzyme assays (e.g.see Lawrence & Niu, 1998, Hanke et al., 1996, Kelloff et al., 1996).

An older, and more extensively studied, class of non-peptide PTKinhibitors is erbstatin and the related tyrphostins (see reviews). Thisclass of inhibitors are active against the receptor PTKs and their modeof inhibition is complex but does not appear to involve binding in thepeptide substrate specificity site regions of the active site (Hsu etal., 1992, Posner et al., 1994). Furthermore, they are inactive againstthe isolated PTK when the unnatural assay metal Mn²⁺ is replaced withthe natural Mg²⁺ (Hsu et al., 1992), are chemically unstable (Budde etal., 1995, Ramdas et al., 1995 & 1994), and are know to be cytotoxic tonormal and neoplastic cells by crosslinking proteins (Stanwell et al.,1995 & 1996) as well as inhibit cell growth by disrupting mitochondriarather than PTK inhibition (Burger et al., 1995).

An important contribution to the protein kinase field has been the x-raystructural work with the serine kinase cAMP-dependent protein kinase(“PKA”) bound to the 20-residue peptide derived from the heat stableinhibitor protein, PKI(5–24), and Mg₂ATP (Taylor et al., 1993). Thisstructural work is particularly valuable because PKA is considered to bea prototype for the entire family of protein kinases since they haveevolved from a single ancestral protein kinase. Sequence alignments ofPKA with other serine and tyrosine kinases have identified a conservedcatalytic core of about 260 residues and 11 highly conserved residueswithin this core (Taylor et al., 1993). Two highly conserved residues ofparticular note for the work proposed herein are the general baseAsp-166 which is proposed to interact with the substrate OH and thepositively charged residue, Lys-168 for serine kinases and an Arg fortyrosine kinases (Knighton et al., 1993), which is proposed to interactwith the γ-phosphate of ATP to help catalyze transfer of this phosphate.Two additional important PKA crystal structures have been reported(Madhusudan et al., 1994), one for the ternary PKA:ADP:PKI(5–24) complexwherein the PKI Ala 21 has been replaced with Ser (thereby becomming asubstrate), and one for the binary PKA:PKI(5–24) complex wherein the PKIAla 21 has been replaced with phosphoserine (an end product inhibitor).The ternary complex shows the serine OH donating a H-bond to Asp-166 andaccepting a H-bond from the side chain of Lys 168. The binary complexshows the phosphate group of phosphoserine forming a salt bridge withthe Lys-168 side chain and within H-bonding distance of the Asp-166carboxyl group. These structures support the earlier proposed roles forAsp-166 and Lys-168 in the catalytic mechanism.

The x-ray structures of PKA show that the enzyme consists of two lobeswherein the smaller lobe binds ATP and the larger lobe the peptidesubstrate. Catalysis occurs at the cleft between the lobes. Thecrystallographic and solution structural studies with PKA have indicatedthat the enzyme undergoes major conformational changes from an “open”form to the “closed” catalytically active form as it binds thesubstrates (Cox et al., 1994). These conformational changes are presumedto involve the closing of the cleft between the two lobes as thesubstrates bind bringing the γ-phosphate of ATP and the Ser OH in closerproximity for direct transfer of the phosphate.

However, the inhibitors of protein kinases still lack the specificityand potency desired for therapeutic use. Due to the key roles played byprotein kinases in a number of different diseases, including cancer,psoriasis, arthrosclerosis, and their role in regulating immune systemactivity, inhibitors of specific protein kinases are needed. The presentinvention provides a novel approach for designing protein kinaseinhibitors, which are more potent as well as being more specific for thetargeted pathways.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying inhibitors ofprotein kinases. A first module having a one or more functional groupsfor binding to catalytic residues of the protein kinase is combined witha second module which provides a non-peptide scaffold. Combinations ofthe first and second modules which inhibit protein kinase activity areselected.

The present invention also provides a method of inhibiting a proteinkinase. The protein kinase is contacted by a compound comprising a firstmodule having a functionality for binding to catalytic residues of theprotein kinase and a second module which provides a non-peptidescaffold. The combination of the first and second modules inhibits theprotein kinase activity.

In a further embodiment, the invention provides a non-peptide proteintyrosine kinase inhibitor having the formula:

wherein R1 is H or OH, R2 is H or OH, R3 is OH or H, R4 is CH₂,CH(CH₃)R, or CH(CH₃)S, R5 is OCH₃, H, or OH, R6 is OCH₃, F, H, or OH,and R7 is OCH₃, H, OH, CO₂H, CO₂CH₃, CH₂CO₂H, or CH₂CO₂CH₃.

The present invention also provides a non-peptide protein tyrosinekinase inhibitor having the formula:

wherein R1 is OH or H, R2 is OH or H, R3 is OH or H, R4 is OH or H, R5is OH, OMe, or H, R6 is OH, OMe, or H, R7 is OH, OMe, or H, and X is 0or 1.

In yet another embodiment, the present invention provides a method oftreating a condition, responsive to a protein kinase inhibitor, in apatient. A protein kinase inhibitor is administered to a patient. Theprotein kinase inhibitor has a first module having a functionality forbinding to catalytic residues of the protein kinase and a second modulewhich provides a non-peptide scaffold. The combination of the first andsecond modules inhibits protein kinase activity in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the modular strategy for developing non-peptide proteinkinase inhibitors. Step 1 utilizes one or more first modules (“M₁'s”) toidentify promising non-peptide scaffolds. Step 2 enhances the potency byadding specificity elements. During this step the scaffolds arevalidated. Whether the inhibitor is non-ATP competitive can also bedetermined. In step 3, the potency and selectivity are further enhancedusing combinatorial libraries to optimize M₁ and specificity elements.

FIG. 2 provides a depiction of the x-ray structure of(PKA):Mg₂ATP:pseudosubstrate inhibitor.

FIG. 3 provides a general module M₁ design features for binding to theconserved protein kinase catalytic region.

FIG. 4 shows that the boronic acid “inhibitors” 21 and 22 were shown tobe substrates for PKA.

FIG. 5 demonstrates the binding interactions of src substrateAc-Ile-Tyr-Gly-Glu-Phe-NH₂ (SEQ. ID. No. 1) in model src active site.

FIG. 6 shows the design of naphthalene-based src inhibitor scaffolds.

FIG. 7 shows the design of isoquinoline and indole-based src inhibitorscaffolds.

FIG. 8 provides an example of the chemistry used to prepare thenaphthalene inhibitors, which is described in Marsilje 2000. A boronicacid functionality can be put put in place of a M₁ hydroxyl groups inthe src inhibitors from Table 5 using the Pd (0)-catalyzedcross-coupling methodology was used wherein either an aryl triflate(Ishiyama et al, 1997) or an aryl halide (Ishiyama, 1995) can be coupledwith the commercially available pinacol ester of diboron.

FIG. 9 shows a synthetic scheme that can be followed, in order to attachhydrophobic S₂ selectivity elements to the naphthalene scaffold.

FIG. 10 shows successful model reactions with naphthalene chemistry,which can be converted to the solid phase in preparation forsynthesizing combinatorial libraries of this scaffold in a 96-well plateformat. The chemistry has been carried out on the less activenaphthalene regioisomer represented by 44 because this compound isreadily obtained from commercially available 3,5-dihydroxy-2-naphthoicacid as describe in Marsilje 2000.

FIG. 11 provides a possible strategy for modifying the naphthalenescaffold in combinatorial libraries.

FIG. 12 shows the conversion of the triflate functionality formed inreaction 2 from intermediate 69 (FIG. 11) to an amine (Wolfe et al,1997) and then a series of amides or other amine derivatives.

FIG. 13 Following the modeling procedure described above, a the seriesof hydroxy-containing analogs of the boronic acid M₁ group shown in FIG.13 were modeled in the src and IRTK (insulin receptor protein tyrosinekinase) active sites and found the illustrated interactions/bindingmodes as some of the interesting possibilities.

FIG. 14 shows results from testing of the non-peptide src inhibitor43-meta (Table V) in the LA25 and NRK cell lines.

FIG. 15A shows a comparison of taxol and doxorubicin (they were moreeffective than etoposide & cisplatin in this tumor cell culture) withthe three Src inhibitors mentioned above utilizing ovarian tumor cellsfrom tumor N015. FIG. 15B shows the results from tests of the srcinhibitors for inhibition of normal human fibroblast cell growth. Noinhibition of normal cell growth (both subconfluent and confluent; someenhanced growth was observed instead) was found, indicating that theseinhibitors are not toxic to normal cells even at a 10-fold higherconcentration. FIG. 15C provides the structures of the src inhibitorsTOM 2-32, TOM 2-47, and KLM 2-31.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for identifying inhibitors ofprotein kinases. The general modular strategy for the development ofnon-peptide PTK inhibitors is outlined in FIG. 1. Basically, a firstmodule having a one or more functional groups for binding to catalyticresidues of the protein kinase is combined with a second module whichprovides a non-peptide scaffold. Combinations of the first and secondmodules which inhibit protein kinase activity are then selected. Step 1begins with protein kinase inhibitor information which was alreadygenerated, i.e. pentapeptide scaffolds which bind in the substratespecificity sites of PKA or src have already been used to positionvarious rationally designed functional groups (i.e. module “M₁” or“first module”) to interact with the conserved catalytic residues, MgATPor MgADP. A selection of preferred functional groups have now beenidentified in this fashion to serve as the initial M₁ module for Step 1.These M₁ functional groups have been utilized to identify promisingnon-peptide scaffolds for src inhibitors in Step 1. It was anticipatedthat these bare non-peptide scaffolds, with only an M₁ appendage, wouldhave low binding affinity and be relatively nonselective among the PTKs.A lack of selectivity at the level of Step 1 is viewed as an advantagefor the development of a general strategy which can be reapplied toadditional PTKs. Therefore the suite of non-peptide scaffolds identifiedin Step 1 can be recycled for use against additional PTKs by rescreeningthem and carrying the better ones through Steps 2 and 3, all using thenew PTK target. The potency of these bare scaffolds from Step 1 may beincreased enough by the attachment of one or two initial specificityelements (S_(n)) to allow for the validation of the scaffold as non-ATPcompetitive and amenable to further potency enhancements usingcombinatorial chemistry in a rationally guided fashion. Promising srcnon-peptide M₂ (second module) scaffolds identified in Step 1 haveundergone Step 2 and displayed a one to two order-of-magnitude increasein potency against src as well as non-competitive binding relative toATP.

Validation of the scaffolds at the level of Step 2 before undertakingthe resource intensive combinatorial library synthesis and testing ofStep 3 is important for three reasons: 1) To develop the chemistry forappending the specificity element (S_(n)) side chains. 2) To determinethat these inhibitors are not ATP-competitive. 3) To determine that thepotency is responding to the side chain S_(n) properties and attachmentpoints as would be expected based upon the working model for thesrc:inhibitor complex (this provides some confidence that rationallyguided choices can be made for the ranges of individual selectivityelements S_(n) to include in the focused libraries of Step 3).

It is in Step 3 that high potency and specificity for a particular PTKis anticipated because numerous combinations of M₁ functional groups(and close analogs M₁′) with selectivity elements (S_(n)) will beevaluated experimentally via combinatorial chemistry and high-throughputscreening. Potency and selectivity may be further increased if necessaryby appending additional specificity elements (see optional S_(n)'s inFIG. 1). One of the selected src inhibitor scaffolds from Step 2 hasalready been attached to a solid phase resin and is currently beingdeveloped into a combinatorial library following Step 3.

In each of the Steps 1–3, molecular modeling studies with theIRTK:peptide:AMP-PNP crystal structure, the model of the src:peptidecomplex and the models for the src complex with the individual familiesof inhibitors based upon a particular scaffold will be used asqualitative guides. These modeling studies have been remarkably helpfulthus far in guiding the inhibitor design efforts as detailed later.Combining structure-based design and combinatorial chemistrytechnologies in this fashion provides a synergy wherein the majorindividual deficiencies of these technologies used in isolation areaddressed by the strengths of the other. The major deficiency ofstructure-based design is the difficulty in quantitatively predictingligand binding affinities, which is particularly challenging due to thecomplex effects of solvation and entropy (Ajay & Murcko, 1995). Themajor strength of structure-based design is its' capability to predictwhat types of molecules are likely to be good ligands. Structure-baseddesign can determine the rough boundaries (proteins have someflexibility which need to be taken into account) for molecular size andshape as well as indicate where hydrophobic, H-bonding and ionicinteractions are likely to occur. On the other hand, the majordeficiency of combinatorial chemistry is that “molecular space” fordrug-sized molecules (i.e. MW ca. 500 or less) is so large that onecould not hope to sample all of this molecular space with a high densityof coverage in a reasonable sized combinatorial library. A recentestimate (Bohacek et al., 1996) of the number of possible compoundscontaining up to 30 atoms chosen only from carbon, nitrogen, oxygen andsulfur (in addition to H's) is 10⁶⁰ compounds. This is in the molecularweight range of typical drug molecules and still does not includeadditional diversity provided by other atoms, e.g. halogens.Consequently, additional constraints need to be used to identify regionsof molecular space wherein particular drug candidates are likely to belocated. Structure-based design can drastically reduce the volume ofmolecular space to be explored by identifying the types of moleculeswhich have a higher probability of being good ligands. The inability toquantitatively predict which of these “focused” combinatorial librarymembers will in fact be the tightest binding ligands (i.e. thequantitation problem) is then resolved by employing an efficientcombinatorial synthesis and high-throughput testing of the library.

In the earlier peptide based serine and tyrosine kinase inhibitor designefforts PKA was used as a convenient qualitative model for designing theprotein kinase inhibitor module M₁ for interaction with the conservedcatalytic residues. There is much more structural and kineticinformation available for PKA than any other protein kinase.

The crystal structure of PKA complexed with Mg₂ATP and a pseudosubstrate(i.e. OH replaced with H) peptide inhibitor (PKI 5-24 amide) has beensolved (Zheng et al., 1993) and the active site interactions near the P0 Ala of this inhibitor are shown in FIG. 2.

This crystal structure shows Mg₂ATP bound to the small lobe of PKA and a20-residue pseudosubstrate peptide inhibitor bound to the large lobewith the overall conformation of the enzyme in the closed (i.e. the twolobes are touching) and activated state. The distances between the P 0Ala side chain carbon and the nearby heavy atoms in the complex areshown in A^(O) in FIG. 1. These distances show that the Ala side chainis within van der Waals contact distance of the surrounding atoms andindicates that there is little space for appending bulky M₁ functionalgroups to the Ala side chain. However, PKA is a flexible enzyme withopen, closed and intermediate conformations (Cox et al., 1994) and thesemore open conformations would result in a retraction back of the ATPγ-phosphate from the inhibitor Ala thereby creating a binding cavity forappended M₁ functional groups. Furthermore, PKA binds MgADP with thesame affinity as MgATP (Whitehouse et al., 1983) and the ratio ofATP/ADP in cells is typically 10/1 (Alberts, et al. 1994). Therefore, atequilibrium, ca. 10% of the cellular protein kinase is in the MgADPbound state and this form of the enzyme can also be targeted with aninhibitor to drain all of the enzyme from the catalytic cycle into aPKA:MgADP:inhibitor inactive complex.

Since the PKA catalytic residues Asp-166 and Lys-168 are completelyconserved in all serine kinases, and the tyrosine kinases only differ bythe substitution of Arg for Lys-168 (Taylor et al., 1993), this regionof the active site was chosen, long with the adjoining MgATP or MgADP,to target a selection of inhibitor functional groups which could serveas M₁ and be broadly useful for developing inhibitors for the entireprotein kinase family. By targeting M₁ to the region of the active siteadjacent to the nucleotide, an orientation point is provided for thenon-peptide inhibitors which can extend into the peptide bindingspecificity sites without always competing with ATP/ADP binding.

A selection of functional groups which could be utilized as M₁ wasidentified first because, although this region of the active site isvery highly conserved, it was expected that each particular proteinkinase will still display some differing preferences across thisselection due to small variations in the active site conformations andadjoining residues. Furthermore, the rank order preference among thisselection of M₁'s may change somewhat as the M₁ module is appended todifferent non-peptide scaffolds. This expectation is based upon thepotential for each non-peptide scaffold to bind in somewhat differentorientations with each individual protein kinase and with eachparticular set of selectivity element (S_(n)) side chains. Pentapeptidescaffolds were chosen for the initial screening of functional groups forM₁ because the binding orientation of these larger peptide scaffolds islikely to be very consistent and predictable (i.e. closely resemblingthat observed by x-ray) throughout the series and could be moreconfidently assumed to position each tested M₁ functionality adjacent tothe conserved catalytic residues as intended. Consequently, the goal ofthis earlier peptide-based work was to identify a collection of M₁functional groups which can be used, not only for the initial screeningof non-peptide scaffolds (Step 1), but also as an initial set of M₁ sidechains which can be further expanded via close analogs and therebyoptimized simultaneously with the other side chains in the finalnon-peptide combinatorial libraries (Step 3).

In order to model the candidate M₁ functional groups in this conservedcatalytic region of the PKA active site, they were built onto the P 0Ala position in the PKA ternary structure using the SYBYL molecularmodeling package (Tripos) on a Silicone Graphics workstation asindicated in FIG. 3.

A crystal structure of PKA with MgATP and an inhibitor bound in a more“open” conformation was not available, so initial modeling studies werecarried out on the MgADP bound form of PKA derived from the ternarycomplex illustrated in FIG. 2 by simply deleting the ATP γ-phosphate.Initial modeling studies were used to provide qualitative guidance foridentifying interesting potential M₁ functional groups for the proteinkinase family before synthesis and testing. The most advancedcomputational algorthms for quantitatively predicting the free energy ofbinding, such as Free Energy Perturbation methods, are computationallyintensive methods which are not practical at this point in time forroutine use by the non-specialist. Even the most advanced methods can beinaccurate due to difficulties in sampling, inadequacies in themolecular mechanics force fields/parameters, and an incompleteunderstanding of electrostatics in water (Ajay & Murcko, 1995). Lessrigorous (and easier to use) computational methods tend to be unreliablein making quantitative predictions of binding affinities, especiallywhen dealing with multiple polar and ionic interactions such as thoseinvolved in M₁ binding.

In order to allow molecular mechanics calculations to be done with theSilicone Graphics workstation in a reasonable amount of time, two layersof residues were carved out from the PKA ternary structure which aresurrounding the PKA active site, along with the peptide inhibitor andMg₂ADP. The M₁ functional groups were then appended to the P 0 Ala sidechain and the entire PKA active site:Mg₂ADP:modified peptide inhibitorcomplex was then subjected to 300 iterations of molecular mechanicsminimization using the Tripos force field with a distance dependentdielectric constant after assigning appropriate formal charges andcalculating Gasteiger Marsili point charges using SYBYL. Setting themaximum number of iterations at 300 was sufficient to remove any seriousstrain in the complexs and yet not allow the overall structure to“drift” significantly from the starting x-ray structure if convergenceis not reached. These minimized complexes were then visually evalulatedto determine if the appended individual M₁ functional groups were ableto engage in favorable interactions with the conserved catalyticresidues and/or Mg₂ADP. This visual evalulation involved, among otherstandard interaction evaluations, measuring atom-atom distances todetermine if hydrogen bonds and ionic interactions were being favorablyformed.

Favorable intermolecular interactions between an individual M₁functionality and the conserved catalytic residues or Mg₂ADP does notnecessarily mean enhance binding affinity will be observed for the newinhibitor. Unfavorable desolvation of both the polar M₁ functionalityand the polar PKA active site residues (as well as complex entropyeffects) are not included in this analysis and may reduce the netbinding affinity to the point that the modified inhibitor may even beless potent that the corresponding P 0 Ala inhibitor, even though theappended M₁ functionality is interacting with the conserved catalyticresidues and/or MgADP (or MgATP) as intended. Even in cases where thisdesolvation penalty results in no net increase in binding affinity,these M₁ functional groups are still useful as an orienting groups forcorrectly positioning the non-peptide inhibitor analogs in the proteinkinase active site. Positioning these polar functional groups elsewherewithin the active site (assuming they are tethered so as not to be ableto extend into bulk solvent while the scaffold is favorably bound in theactive site) is likely to result in a reduced binding affinity becausethey were specifically designed and selected based upon theirdemonstrated ability (while appropriately tethered to pentapeptidescaffolds) to be accepted adjacent to the conserved catalytic residuesand MgADP/MgATP. If a particular M₁ functionality does not correctlyposition a non-peptide scaffold in Step 1 then attempts to improve thepotency by rationally appending initial specificity elements in Step 2would likely fail.

None of the literature protein kinase assay procedures contain addedADP. A typical PKA literature assay procedure (Glass et al., 1989) wasmodified by adding 10% as much ADP as the ATP concentration used toreflect the natural 1/10 ratio in the cell. This protein kinase assay ishereinafter referred to as the “Literature Mimetic” assay. It has beenused for PKA as well as the src. An examination of the literature, andcommercially available protein kinase assays, showed that there is poorconsistency from lab to lab and company to company and that all of theseassays use physical chemical conditions which differ considerably fromthose known to exist inside cells. Since inhibition of intracellularprotein kinases is the ultimate goal for drug discovery, new proteinkinase assays have been developed which come much closer to mimickingthe overall cytosolic physical chemical conditions known to exist insidecells. The development of these “Cellular Mimetic” protein kinaseassays, is described herein, along with a novel method for determiningwhich form of a protein kinase a given inhibitor binds best to (theSTAIRe method). Data was collected correlating the activity of the newnon-peptide src inhibitors in the Cellular Mimetic assay with thatobtained in the LA25 src transformed cell line (see below).

When these two assay conditions were applied to some of thepentapeptide-based PKA inhibitors, which were designed as illustrated inFIG. 3, the results shown in Table 1 were obtained. The same assayconditions were also applied to the analogously designedpentapeptide-based src inhibitors and obtained the results shown inTable 2.

The standard pentapeptide sequence chosen for the majority of PKAinhibitors in Table 1 was derived from the pseudosubstrate sequence ofthe peptide inhibitor which was bound to PKA, when the crystal structureillustrated in FIG. 1 was solved. The standard pentapeptide sequenceused for src in Table 2, Ac-Ile-Xaa-Gly-Glu-Phe-NH₂ (SEQ. ID. No. 2),was described in Nair, Kim et al., 1995. Some of the chemistry used toprepare the PKA inhibitors is described in Nair, Lee & Hangauer 1995.The synthetic methodology used to develop a number of the src inhibitorsis described in Lai et al., 1998.

The collective results in Tables 1 and 2 show that both the serinekinase PKA and the PTK src can accommodate a variety of large polar M₁functional groups at the P 0 phosphorylation position. Furthermore,using the STAIRe methodology (see Choi et al. 1996), the sulfamic acidinhibitor 8, and related inhibitors, were shown to actually bind bestwhen MgATP (not MgADP or no nucleotide) is also bound. This was asomewhat surprising result since these inhibitors are analogs of the“end product inhibitors” 1 & 12 which must bind simultaneously withMgADP just following phosphate transfer in the generally acceptedreaction mechanism for protein kinases.

These results also demonstrate that both PKA and src can show a largedifference in binding affinity for structurally very similar inhibitors.For example, the sulfamic acid PKA inhibitor 8 (Table I) has a K_(i) of0.16 μM under Literature Mimetic assay conditions (L) whereas theisosteric sulfonamide 7 is 1,875× less potent (K_(i)=300 μM). Thesulfamic acid inhibitor 8 is also isosteric with the end productphosphate inhibitor 1 yet it binds much more tightly under bothLiterature Mimetic assay conditions (31X) and Cellular Mimetic (C) assayconditions (108X). The beneficial effect of an oxygen atom positionedanalogously to that in the substrate Ser is illustrated by comparison ofphosphonate 2 to phosphate 1 and also ether 6 to phosphate 1. Thisoxygen atom can also be positioned as a serine-like OH side chain andenhance binding (compare 2 to 3A and 4A) wherein the closer serine mimic4A is the more active. The difference in activity of the diasteromericinhibitors 3A or B and 4A or B suggests a specific interaction with theactive site catalytic residue Asp-166 may in fact be occurring asintended in the M₁ design (FIG. 3).

TABLE I INITIAL M₁ SCREENING RESULTS WHILE APPENDED TO THE PKAPENTAPEPTIDE SCAFFOLD

$\frac{{K_{i}\left( {\mu\; M} \right)},\left( {Conditions}^{*} \right)}{\begin{matrix}{\;^{*}L = {{Literature}\mspace{14mu}{Mimetic}}} \\{C = {{Cellular}\mspace{14mu}{Mimetic}}}\end{matrix}}$ 1

2

76 (L) NT (C) 3

18 (L)-Diastereomer A72 (L)-Diastereomer B NT (C) 4

5   6 X = H   X = CO₂H

7

8

9

10

11

45 (L) NT (C) ---- = Attachment Point

The structure identified in Table 1 as Ac-Arg-Arg-Gly-Ala bonded toM₁-Ile-NH₂ is SEQ ID NO. 2.

The src inhibition results (Table II) show that the end productinhibitor 12 drops in activity upon going from Literature Mimetic assayconditions to the higher ionic strength Cellular Mimetic assayconditions analogous to the PKA end product inhibitor 1. However,whereas all of the PKA inhibitors with polar M₁ functional groups wereless active under Cellular Mimetic assay conditions three of the srcinhibitors 14, 15, & 17 held their activity under these higher ionicstrength assay conditions. Also, the hydroxyphosphonate src inhibitor 13(a mixture of the R and S diastereomers) is analogous to the PKAinhibitor 3A and both are roughly in the same activity range as theircorresponding end product inhibitors, 12 & 1 respectively, underLiterature Mimetic assay conditions. Shortening the side chain length inthe phosphonate src inhibitor 13 by one carbon atom (and necessarilyremoving the attached OH at the same time) to give 14 improved theactivity (analogous to the PKA inhibitor comparison 3 to 4) and, moreimportantly, resulted in equivalent activity under Cellular Mimeticassay conditions. The src results with 16–19 (particularly 17, see laterfor an analogous α-tricarbonyl acid M₁ analog appended to non-peptidesrc inhibitors) also suggests that similar amides may be useful M₁functional groups to explore with non-peptide src inhibitors.

Non-peptide src inhibitors are preferred to peptide scaffold basedcompounds, partly because some of these inhibitors have a dual effect onsrc. For example, phosphonate inhibitor 14 not only inhibits src bycompetitively binding in the active site but it also activates src bybinding to the SH2 site thereby releasing the intramolecularautoinhibition mechanism (Xu et al., 1997). This opposing effect givesan unusual IC₅₀ curve for 14 wherein at low inhibitor concentrations srcis stimulated (to a maximum of 70%) in a smooth dose-response fashion(due to initial tighter SH2 binding) followed by a typical IC₅₀inhibition curve at higher inhibitor concentrations (due to loweraffinity blockade of the active site). This opposing activation effectof the pentapeptide inhibitors makes them appear to be less potentactive site inhibitors than they in fact are, and makes it difficult toaccurately rank M₁ groups while appended to this pentapeptide scaffold.However, the better M₁ groups identified with the src pentapeptidescaffold must still be accommodated in the catalytic region of theactive site and hence are useful orienting groups for the ongoingnon-pepitde src inhibitor studies as intended. Since PKA does not havean SH2 domain this complication is not a factor in interpreting the PKApentapeptide inhibitor M₁ testing data.

TABLE II INITIAL M₁ SCREENING RESULTS WHILE APPENDED TO THE SRCPENTAPEPTIDE SCAFFOLD

% Inhibition of 2 mM RR-src phosphorylation by src Assay ConditionsLiterature Inhibitor (1 mM) Mimetic Cellular Mimetic 12

36 0 13

51 0 14

83 88 15

68 59 16

60 8 17

20 28 18

64 5 19

24 0

The structure identified in Table II as Ac-Ile-Tyr bonded toM₁-Gly-Glu-Phe-NH₂ is SEQ. ID. No. 2.

The results in Tables 1 and 2 also show how much effect the assayconditions can have on both inhibitor potencies and the rank order ofactivity. For example, as shown in Table 1, switching from theLiterature Mimetic (L) assay conditions to the Cellular Mimetic (C)assay conditions can change the potency from as little as 3-fold(inhibitor 10) to as much as 108-fold (inhibitor 1). Also, whereasinhibitor 10 is less potent than 1 under Literature Mimetic conditionsit is more potent under Cellular Mimetic conditions. The src inhibitordata presented in Table 2 show that many of the inhibitors lose theirpotency upon going from Literature Mimetic assay conditions to CellularMimetic assay conditions. The rank order of potency against src is alsosensitive to the assay conditions. Whereas inhibitor 18 is more potentthan inhibitor 17 under Literature Mimetic conditions, the opposite istrue under Cellular Mimetic conditions. Since activity within cells isthe goal, the Cellular Mimetic src assay was selected as the standardassay for testing potential non-peptide src inhibitors. Activity withinthe Cellular Mimetic assay is a necessary, but not sufficient, conditionfor activity within cells. As will be described later, the CellularMimetic src assay will be followed up with cell culture assays whereincell penetration, metabolism and binding to other cellular componentsare also factors in the measured potency.

The next class of M₁ functionality which was explored was the boronicacid group. This functional group is an intriguing candidate for M₁ fora number of reasons: 1) It can exist in a non-ionic state so that itshould not prevent passive absorption of non-peptide inhibitors acrosscell membranes. 2) The planar, trigonal, boron acids might formreversible tetrahedral covalent borate complexes (a well known propertyof boronic acids, see Loomis & Durst, 1992) through their vacant 2porbitals with anions present in the protein kinase active site such asthe catalytic Asp carboxyl group or the ATP/ADP terminal phosphateoxygens. This ability to form borate complexes with active sitenucleophiles has been extensively utilized to develop slow bindinginhibitors of serine proteases (e.g. see Kettner & Shenvi, 1984),wherein the nucleophilic serine OH forms a covalent bond with the vacant2p orbital in the boronic acid resulting in a tetrahedral borate complex(e.g. see Skordalakes et al., 1997). Also, an intramolecular complex ofa boronic acid with a urea NH₂ was used to prepare transition stateanalogs inhibitors of dihydroorotase (Kinder et al., 1990). 3) Boronicacids act as Lewis acids and are converted to tetrahedral hydrates inwater by forming borate complexes with water or hydroxide ions.Therefore, it is also possible that these boronic acid hydrates mayfunction as phosphate mimics and M₁ modules as proposed in FIG. 2. Thishydration property was utilized by Baggio et al. (1997) wherein ahydrated boronic acid functioned as a transition state analog inhibitorfunctionality for arginase. These researchers evaluated the inhibitedcomplex by x-ray and showed that the hydrated boronic acid functionalityformed two hydrogen bonds with the active site catalytic Glu-277carboxyl side chain and one of the other hydrated boronic acid OH'sinteracted with two catalytic Mn²⁺'s in the active site. These bindinginteractions are closely analogous to those proposed in protein kinaseactive sites, i.e. H-bonds to the catalytic Asp side chain carboxylgroup and interactions with the active site Mg²⁺'s (see FIG. 2), and 4)The use of boronic acids for protein kinase inhibitors has not beenexplored previously.

In the area of pentapeptide-based PKA inhibitors, the boronic acidfunctionality has been prepared and tested as a potential M₁ moduleutilizing the four inhibitors 21–24 shown in Table 3 (see Hsiao &Hangauer, 1998, for some of the chemistry used to prepare thesecompounds).

TABLE III PKA INHIBITION RESULTS WITH BORONIC ACID-CONTAINING PEPTIDEINHIBITORS IC₅₀ μM (cond. IC₅₀ μM (cond. IC₅₀ μM (cond. IC₅₀ μM (cond.Ac-RRGXI- L, 0 h L, 4 h C, 0 h C, 4 h NH₂, X = preincubation)preincubation) preincubation) preincubation) 20 Ala 278 (K_(i) = 9 μM)417 41 (K_(i) = 25 μM) 50 21

249 *500 μM34% inh  764 *2000 μM19% sti 22

 81 *65 *1753 *2000 μM71% sti 23

398 133 2000 μM16% inh *2000 μM5% inh 24

1000 μM33% inh 1000 μM44% inh 2000 μM6% sti 1734 μM *Very distorted IC₅₀curve: Suggests Inhibitor is also a substrate. L = Literature MimeticAssay Conditions. C = Cellular Mimetic Assay Conditions. Inh =Inhibition. Sti = Stimulation.

The structure identified in Table III as Ac-RRGXI-NH₂ is SEQ. ID. No. 4.

While testing these boronic acid-containing PKA inhibitors, thecorresponding pentapeptide pseudosubstrate inhibitor 20 was included asan internal control while investigating time-dependent inhibition asshown in Table 3. Under Literature Mimetic assay conditions, and nopreincubation, the initial results suggested that the shortest chainL-amino acid 21 was binding with the same affinity as thepseudosubstrate inhibitor 20 (i.e. K_(i) ca. 9 μM). As this side chainwas increased in length (to 23 and then 24) binding affinity appeared todecrease. When the stereochemistry of the unnatural amino acid wasinverted from L in 21 to D in 22, binding affinity appeared to increase3-fold. This improvement in binding may occur as a result that theboronic acid OH in 21 is positioned at the same chain length asL-homoserine whereas the natural substrate, L-serine, has a one carbonshorter side chain. Modeling results with the PKA ternary structureindicated that the boronic acid OH can be retracted back somewhat byinverting the α-carbon stereochemistry from L in 21 to D in 22 and thenrepositioning the side chain to more closely mimic the positioning ofthe natural substrate L-serine OH adjacent to the catalytic residues(Asp-166 and Arg-168). The modeling results were subsequently supportedby the finding that, upon incubation of PKA with these inhibitors for upto 4 hours without adding the competing peptide substrate (Kemptamide:LRRASLG-NH₂) (SEQ. ID. No. 5), both 21 and 22 function as substrateswith he D-diastereomer 22 being phosphorylated faster.

The fact that these boronic acid inhibitors are also substrates, becamemuch more obvious by the greatly distorted IC₅₀ curves obtained underthe Cellular Mimetic conditions, both with and without preincubation(both PKA and src are more active enzymes under the Cellular Mimeticconditions than under Literature Mimetic conditions). In the assay usedto obtain these results, the p³² phosphorylated Kemptamide product (25generated from γ-P³² ATP) was isolated at the end of the substrateincubation period by binding to phosphocellulose filter paper via thethree cationic groups (two Arg's and the N-terminus) and the level ofphosphorylated product isolated on the paper is then measured by liquidscintillation counting (cpm's). The boronic acid inhibitors 21–24 havetwo Arg's in their sequence also and therefore will bind to thephosphocellulose paper in addition to Kemptamide (although not asconsistently or completely due to one less positive charge).Consequently, when analyzed as inhibitors, the amount of phosphorylatedKemptamide produced was not only counted, but also the amount ofphosphorylated inhibitor simultaneously produced (e.g. see 26 below).The net result is that distorted IC₅₀ curves are obtained which show net“stimulation” at higher inhibitor concentrations in some cases. The Ddiastereomer 22 gives the greatest apparent “stimulation” (71%) whenpreincubated with PKA for 4 hours under Cellular Mimetic conditionsfollowed by the L diastereomer 21 (19%) and then the one carbon homolog23 (5%) indicating all three are substrates for PKA (Table III). Theunderlying substrate behavior of these “inhibitors” makes an accuratemeasurement of their inhibition potency impossible with the currentassay. However, it does appear from the data that homologating theboronic acid functionality out with only CH₂ groups (homologations withboronic acid non-peptide src inhibitors may also be carried out)decreases the binding affinity and ability to function as a substrate.

Phosphorylated 22 is SEQ. ID. No. 4. The boronic acid “inhibitors” 21and 22 were shown to be substrates for PKA running the same assay, butwithout adding Kemptamide, and stopping the reaction at various timepoints as shown in FIG. 4. The graphs show their respective rates andlevels of phosphorylation with the typical loss of initial velocitykinetics with time (due to substrate depletion and end productinhibition), analogous to a standard L-Ser substrate such as Kemptamide.The comparison of 21 to 22 shown was done in the same assay run, atidentical boronic acid substrate concentrations, and with identicalCellular Mimetic assay solutions so that the cpm's could be directlycompared. The graphs show that initial velocity conditions were lostwithin one hour for D isomer 22 whereas the linearity appears to havebeen lost somewhat slower with the L isomer 21 suggesting a slowerconsumption of starting material. That the boronic acid moiety would bephosphorylated by PKA was surprising, but it is even more surprisingthat the phosphonic-boronic acid mixed anhydride produced (e.g. 26) wasstable enough to survive the pH 7.2/37° C. assay incubation and then beisolated by binding to phosphocellulose paper after acid quenching ofthe reaction with 10% TCA and washing the phosphocellulose paper with 25mM phosphoric acid (3X). An STN substructure search was run on mixedanhydrides of phosphoric and boronic acids and found only threereferences to experiments and theoretical calculations for the analogousputative (but not proven) anhydride formed from boric acid andphosphoric acid as a solid surface impregnated catalyst for the partialoxidation of ethane to acetaldehyde at 823° K. (Zhanpeisov & Otsuka,1992, Otsuka et al., 1992, Murakami et al., 1990). However, this highlyunusual anhydride has never before been synthesized free of a solidsurface, isolated or characterized. Thus, this is a novel enzymaticreaction and chemical entity with interesting possibilities for proteinkinase inhibitor designs.

The src and PKA pentapeptide scaffold tethered M₁ evalulations describedabove have resulted in identifying a variety of orienting M₁ groupswhich could be used for screening potential non-peptide scaffolds asindicated in Step 1 (FIG. 1). The boronic acid (from 22), thephosphonate (from 14), and the sulfamic acid (from 8) were initiallychosen from the menu of potential M₁'s for the src non-peptide scaffoldscreening. Among these choices, the boronic acid M₁ group has proveneffective for Step 1 screening of non-peptide scaffolds.

The most useful crystal structures available for the design ofnon-peptide src inhibitors, which do not compete with ATP, are thenative src structure and the IRTK:peptide:AMP-PNP ternary structure. Forall of the modeling studies discussed below, the SYBYL molecularmodeling software package is used on a Silicone Graphics Workstation.

Since the src and IRTK structures are only used as qualitative guides indesigning the non-peptide scaffolds and combinatorial libraries, theactive sites along with two layers of surrounding residues were carvedout from the native src and IRTK ternary structures, analogous to theprevious PKA modeling studies. The IRTK:peptide:AMP-PNP ternarystructure active site region was used as the template structure to guidethe building of the src residue sequence 424–418 back onto the srcstructure using the comparative homology modeling technique (seeHutchins & Greer, 1991). These residues were disordered in the nativesrc crystal structure and therefore not visible by x-ray. They werereintroduced because they help form the P+1 to P+3 binding sites forpeptide substrates which are important for some of the modeling studies.The analogous residues in the IRTK ternary structure are seen by x-rayand directly interact with the bound peptide substrate. In fact, it isprobably the presence of the bound peptide substrate which induces orderin the positioning of this sequence so that it is visible by x-ray. Thesrc pentapeptide substrate Ac-Ile-Tyr-Gly-Glu-Phe-NH₂ (SEQ. ID No. 1)(Nair et al., 1995) was then docked into the src active site again usingthe IRTK ternary structure as a template. Small adjustments were thenmanually made to partially clean up this complex, all of the hydrogenatoms were added, appropriate formal and partial charges (calculated viathe Gasteiger Marsili method) were added, and then the entire complexwas subjected to 300 iterations of molecular mechanics minimizationusing the Tripos force field, analogous to the previous PKA modelingprocedure. A schematic representation of this modeled complex is givenin FIG. 5. Any inaccuracies in this src:peptide and the src:inhibitormodels are accommodated by experimentally evaluating a range of sidechains, the number and diversity of which is scaled roughly to the levelof uncertainty for the structure of their particular binding region inthe src model active site (see later), in a combinatorial fashion.

As shown in FIG. 5 the residues 424–418 built back into the src interactwith the P+1 to P+3 substrate residues, Gly-Glu-Phe-NH₂ respectively,through beta sheet type hydrogen bonding interactions with the substratemain chain (analogous to the IRTK peptide substrate). Lys 423 engages intwo important interactions: 1) the β and γ CH₂'s fold over the top ofthe P O Tyr phenyl ring engaging in a hydrophobic binding interactionand then 2) the remaining CH₂—CH₂—NH₃ ⁺ of this side chain extends awayto form a salt bridge with the P+2 Glu side chain as indicated. The restof the P 0 Tyr hydrophobic binding pocket is formed by Pro 425 under thephenyl ring and part of the Cys 277 side chain above the phenyl ring.Using a large combinatorial peptide src substrate library, Songyang etal. (1995) found that the most commonly chosen side chain for the P+1position was Gly followed by Glu. The present model indicates that a P+1Glu side chain may form a salt bridge with nearby Arg 469 as indicatedin FIG. 5. Previously, researchers found that only Glu was chosen forthe P+2 position and the present model indicates that this side chainforms a salt bridge with the Lys 423 side chain. At the P+3 position Phewas very strongly preferred and the model indicates that this side chainforms a stacking interaction with the Phe 424 side chain. At the P−1position Songyang et al. found that Ile was the most preferred residuefollowed by Val and then Leu. The model shows a hydrophobic pocket forbinding the P−1 side chain formed mainly by Trp 428, Ala 390 and Leu347. One might expect that the P 0 Tyr side main chain will stronglyinteract (though hydrogen bonding) with the active site in acatalytically competent complex because enzymes often form more criticalinteractions in this region close to where the reaction will beoccurring. The IRTK ternary complex does not show a good hydrogen bondto either the P 0 Tyr NH or carbonyl. The nearest candidate residue forthis interaction in the IRTK structure is Asn 1215 wherein the sidechain NH₂ is 3.71 A^(o) from the Tyr carbonyl oxygen. When the IRTKternary structure is overlayed onto the src native structure, using thefour residues mentioned in the Background and Significance section, Asn468 from the src structure was found to be positioned very close to theanalogous IRTK Asn 1215. This suggests that this conserved residue isperforming an important role and might move a little closer (i.e. about1 A^(o)) to the substrate P 0 NH and carbonyl in a catalytically activecomplex and form the hydrogen bonding interactions indicated in FIG. 5.Finally, the catalytic Arg 388 and Asp 386 are correctly positioned inthe src model to catalyze the transfer of the γ-phosphate from ATP tothe Tyr OH.

The src:peptide substrate complex can now be used to model potentialnon-peptide scaffolds and determine preferred substitution positions forthe specificity elements, all with an appropriately attached M₁functionality, before choosing new scaffolds to experimentally evaluate.The IRTK:peptide:AMP-PNP ternary structure can also be used to modelthese potential scaffolds and preferred substitution positions. Thesescaffolds have broad utility for the development of selective PTKinhibitors by further developing them with appropriate specificityelements following the strategy outlined in FIG. 1.

The first non-peptide scaffold evaluated with this src:peptide substratemodel was the naphthalene scaffold. This is the first use of bicyclicaromatic scaffolds for non-peptide PTK inhibitors, which do not competewith ATP. The naphthalene scaffold's utility for this purpose wasdemonstrated by developing a non-peptide inhibitor of the IRTK and EGFreceptor PTK (Saperstein et al., 1989). The IRTK ternary complexes weresubsequently used to adapt this scaffold for src inhibition (seeMarsilje et al., 2000). The naphthalene scaffold was docked into the srcactive site by first carrying out a least squares fitting of atoms a–donto the peptide substrate as indicated in FIG. 6. In this way thenaphthalene scaffold is related to the peptide substrate by thecyclization shown by the arrow in FIG. 6 and an appended OH as asubstitute for the substrate Tyr NH. This is essentially the sameprocess used to dock this scaffold into the IRTK structure as describedin Marsilje 2000. The peptide substrate was then deleted from the activesite, various M₁ functional groups and specificity elements S₂ & S₃ werethen added to the scaffold as indicated and the complexes were thenindividually minimized for 300 iterations. This same process was alsoused to design the isoquinoline and indole scaffolds whose binding modesare indicated in FIG. 7.

In all of these modeled complexes selectivity element S₂ consists ofvarious hydrophobic side chains which can bind in the same pocket as thesubstrate P−1 Ile side chain and selectivity element S₃ consists ofvarious molecular fragments which can bind in the P+1 to P+3 region ofthe peptide substrate binding sites (FIG. 5). Since the active siteregion where M₁ binds is highly conserved among all of the proteinkinases, the small menu of M₁ functional groups previously identifiedusing peptide scaffolds served as the initial M₁ groups for attachmentto the scaffolds at the indicated positions. Of the two selectivityelements binding sites, the structure of the hydrophobic binding cavityfor S₂ is known with greater confidence in the src model than is the P+1to P+3 binding region for S₃. This is because the S₃ binding site wasconstructed partially by comparative homology modeling whereas the S₂site is largely unchanged from the structure determined by x-ray fornative src. In view of these varied levels of confidence in the modeledbinding sites for M₁, S₂ and S₃, the combinatorial library diversity isscaled such that the greatest variety and number of side chains in thecombinatorial libraries are at the S₃ site followed by the S₂ site andthen M₁.

The src results using M₁ functional groups to experimentally identifypromising non-peptide scaffolds are listed in Table 4. The data in Table4 allows a number of conclusions to be drawn: 1) Low, but measurable,inhibition potency can be obtained with an appropriate M₁ group attachedto a scaffold (e.g. 27 & 38). 2) 1 mM inhibitor concentrations for thistype of screening is higher than desirable but 100 μM is too low.Screening of scaffolds bearing an M₁ group would optimally be conductedat 500 μM. 3) The boronic acid, sulfamic acid and phosphonic acid M₁functional groups, which had been identified using the PKA pentapeptidescaffold (22, Table 3 & 8 Table 1) or the src pentapeptide scaffold (14,Table 2), respectively, give measurable activity when placed at the 2position of the naphthalene ring (27, 28 & 30, respectively), thepreferred position for M₁ identified in the model naphthaleneinhibitor:src complex (FIG. 6). Moving the boronic acid or phosphonicacid M₁ groups to the 1 position (32 or 33) reduced activity. 4) Therelated M₁ sulfonamide functionality, which was poor on the PKApentapeptide scaffold (7 & 9, Table 1) is also poor when appended to the2 (31) or 1 (34) position of the naphthalene scaffold. The sulfonic acidanalog at the naphthalene 2 position (29) is completely inactive, evenat 1 mM. 5) The naphthalene scaffold can be replaced with a benzofuran(35) or a benzothiophene (36) scaffold without a noticeable reduction inactivity when the boronic acid M₁ group is positioned analogous to the 2position on a naphthalene. 6) The boronic acid M₁ group also providesactive compounds when appended to the 26 isoquinoline (37) or indole(38) scaffolds at the positions indicated by modeling results (FIG. 7).However, the indole scaffold is clearly favored over the isoquinolinescaffold suggesting that a hydrogen bond donating ability to Asn 468(see FIG. 7) is important for higher activity (this would require theprotonated isoquinoline which is difavored by the adjacent electronwithdrawing ester group). This conclusion is also supported byconsidering that a peptide substrate may position a hydrogen bonddonating peptide bond NH at a similar position (FIG. 6) and by findingthat an equivalently positioned phenolic OH (FIG. 6) improves potency(phenolic OH's are much better H-bond donors than acceptors) 8) Whendirectly compared to other M₁ groups the boronic acid group is superior(e.g. 27 vs. 28–31, 38 vs. 39). 9) A biphenyl scaffold modeled into thesrc and IRTK active sites and found promising binding modes for thisscaffold. Combinatorial libraries were developed with the biphenylscaffold (see Pavia et al., 1996), and the modeling results wereencouraging. Therefore, the para (40) and meta (41) isomers wereevaluated with the boronic acid M₁ group. Both biphenyl compounds showedpotency equivalent to the best naphthalene boronic acid (27) andtherefore provide another scaffold geometry (the two phenyl rings arenot planar) for further evaluation and development.

Since the bare scaffolds, with only an M₁ group appended, often have lowbinding affinity, the IC₅₀'s and K_(i)'s for the 2-naphthalene boronicacid and sulfamic acid inhibitors were determined to ensure that atypical dose/response IC₅₀ curve is obtained. This analysis provided thetypical shape dose/response curves seen with more potent inhibitors. TheIC₅₀'s and K_(i)'s of these simple inhibitors also confirmed that theboronic acid inhibitor 27 is the more potent than the sulfamic acidanalog 28 and has a K_(i) of about 554 μM.

The next issue addressed with these simple inhibitors before proceedingto elaborate them further was their mode of inhibition, specificallywhether they are ATP-competitive inhibitors. In the case of thenaphthalene inhibitors 27 & 28, their IC₅₀'s were monitored as the ATPconcentration was increased in three steps up to 1 mM. As a comparison,the IC₅₀ of the pentapeptide phosphonic acid src inhibitor 14 (Table II)was also monitored. If any of these inhibitors were competing with ATP,then their IC₅₀'s should have increased proportionally with the ATPconcentration (i.e. the dashed line). As shown, the IC₅₀'s for all threeinhibitors remained essentially constant as the ATP concentration wasincreased demonstrating that they are not ATP-competitive inhibitors. Avery similar, but much less costly (commercial src is expensive),analysis was conducted with the indole boronic acid inhibitor 38. Inthis case, the % inhibition was monitored with 38 at a constant 500 μMinhibitor concentration but with increasing ATP concentrations of 200,500 and 1,000 μM. Once again the inhibitor potency was not reduced bythe increasing ATP concentration demonstrating that 38 is also non-ATPcompetitive.

The initial results obtained in Step 1 suggests that it is possible toidentify promising scaffolds for further elaboration with thisprocedure. The biggest uncertainty with Step 1 is that some of thescaffolds identified in this way might not be binding in the fashionsuggested by the prior modeling evaluations. This is essentially a“false positive” problem. These “false positives” will likely fail inStep 2, when they are evaluated for improved binding using the modeledcomplexes as a guide. Some false positive results can be accepted inStep 1 because the bare scaffolds with only the M₁ group attached areeasily obtained. For further inhibitor development, one may return toStep 1 each time new scaffolds are needed to carry through Steps 2 & 3.The best M₁ generated can be used each time Step 1 is repeated.Currently, the boronic acid M₁ group is preferred since it has a provenability to give measurable activity with bare scaffolds. Also theboronic acid M₁ group offers multiple interesting possibilities forcovalent and non-covalent interactions with the conserved catalyticresidues since it can: 1) hydrate, 2) form borate complexes withelectron rich active site atoms, and/or 3) be phosphorylated and thenreact with active site nucleophiles or engage in additional non-covalentinteractions. From the data in Table 4, the naphthalene and indolescaffolds were chosen as M₂ for the first efforts in Step 2 (thebiphenyl scaffold is also a preferred scaffold). It is also worthmentioning that naphthylalanine and analogs can be successfullysubstituted for the P 0 tyrosine in src peptide substrates (e.g. seeAlfaro-Lopez et al., 1998) further supporting the notion thatnaphthalene and related scaffolds can bind at the P 0 site.

TABLE IV INITIAL STEP 1 RESULTS % SRC INHIBITION IN CELLULAR MIMETICASSAY % Inhibition of 2 mM RR-src at Inhibitor Inhibitor Concentration ()

27

59 (1 mM)13 (100 μM)IC₅₀ = 950 μMK_(i) = 554 μM NON-ATPCOM-PETITIVE 28

31 (1 mM)IC₅₀ = 1.6 mMK_(i) = 963 μM NON-ATPCOM-PETITIVE 29

0 (1 mM) 30

14 (1 mM) 31

0 (100 μM)

32

0 (100 μM) 33

1 (1 mM) 34

0 (100 μM) 35

10 (100 μM) 36 X = S 12 (100 μM) 37

13 (500 μM)

38

NON-ATPCOM-PETITIVE 62 (500 μM) 39

11 (500 μM) 40

13 (100 μM) 41

14 (100 μM) --- = Attaching bond.

In comparing the naphthalene vs. indole scaffold results with a boronicacid M₁ group (i.e. 27 vs. 38, Table 4) the indole hydrogen bonddonating NH and/or the adjacent ester group appeared to be the reasonfor the enhanced potency. Consequently, for Step 2 one of the firstattempts was to add a hydroxyl group and an amide (with S₂) to thenaphthalene scaffold at the adjacent positions suggested by the modelingresults (FIG. 6). For the indole scaffold one priority was to preparesome amide analogs to see if potency can be increased with the S₂specificity element (FIG. 7). In order to facilitate the synthesis ofthese initial analogs, an OH was temporarily substituted for the boronicacid M₁ group. The OH group is also known to interact with the catalyticresidues, as required for an M₁ group, because it is the naturalsubstrate M₁ whose phosphorylation rate is accelerated by interactionswith the catalytic residues. The results obtained for some of theinitial analogs are given in Table 5 along with a side by sidecomparison, in the Cellular Mimetic src assay, to two literature srcinhibitors 50 & 51 which are reported be non-ATP competitive. Some ofthese results and additional analogs are described in Marsilje 2000.

Inhibitor 50, and analogs (Huang et al., 1995), were of particularinterest because the iminochromene scaffold is closely related to thenaphthalene scaffold and it's binding mode would be expected to be verysimilar based upon the model (FIG. 6). Partly because of this closeanalogy, the amides of hydroxyanilines with the naphthalene and indolescaffolds were examined as shown in Table 5. Also, the modeling studieswith these hydroxyaniline amides derivatives in the src active siteindicated that the hydroxyl group may be able to engage in a hydrogenbonding interactions with the src Phe 424-Ala 422 backbone peptide bondsanalogous to peptide substrates (see FIG. 5). These modeling studiesalso indicated that the homologous hydroxybenzylamides should be activeand, more importantly, provide a substitution position (i.e. thebenzylic carbon) for appending side chains to bind in the P−1 side chainpocket (e.g. to Arg 469, FIG. 5).

The data in Table 5 allow the following conclusions to be drawn: 1)Adding an amide extension onto both the naphthalene and indole scaffoldscan increase potency as predicted by the models for these scaffoldsbound in the src active site (ca. 5-fold in the cases of 42 vs. 43-meta& 47 vs 48). 2) Adding a hydroxyl group to the naphthalene scaffoldadjacent to the amide increases potency (about 5-fold, 43-meta vs. 44)as predicted by the src model, and also suggests Asn 468 does hydrogenbond with this OH. 3) Moving the M₁ OH group from the position predictedto be best in the src model to the adjacent position reduces potency byone order of magnitude (43-meta to 45). 4) The indole scaffold is lessresponsive than the naphthalene scaffold to regiochemistry of thehydroxyaniline extension (48 vs. 43). 5) The naphthalene and the indolescaffolds accept the one carbon homologation provide by usinghydroxybenzylamides (46 vs. 43 & 49 vs. 48). 6) The two M₁ hydroxyregioisomers of the naphthalene scaffold are both non-ATP competitive(see Marsilje 2000). 7) All of the methyl hydroxyaniline andhydroxybenzylamide inhibitors were found to be less active suggestingthat the hydroxyl group in the amide extension is functioning as ahydrogen bond donor. In this regard it is worth mentioning that inanother src peptide substrate combinatorial library study Ser and Thrwere identified as two of the most preferred residues at the P+2position (Alfaro-Lopez et al., 1998) suggesting that there are otherbinding opportunities for an amide extension OH other than to thePhe424-Ala 422 peptide bonds suggested by the modeling studies. 8) Themost potent non-ATP competitive, non-peptide, src inhibitor previouslydisclosed in the literature (50) is not nearly as potent as reportedwhen tested under the Cellular Mimetic assay conditions (IC₅₀=118 nMreported by Huang et al., 1995 vs only 30% inhibition at 100 μM) and isless potent than a number of the current inhibitors (especially 43-meta)including the most analogous inhibitor (50 vs. 45). Thestructure-activity-relationship (SAR) reported for hydroxy regioisomersof 50 in their assay (Huang et al., 1995) also does not correspond withthe SAR which was obtained for the related naphthalene inhibitors. Forexample, their iminochromene analog of the most potent naphthaleneinhibitor 43-meta is 230-fold less potent than 50 in their src assay. Animportant advantage of the naphthalene scaffold over the iminochromenescaffold is that it allows a highly desirable S₂ specificity element tobe added for accessing the P−1 hydrophobic site (see FIG. 6) whereas theanalogous position can not be substituted on the iminochromene scaffoldbecause it is occupied by the ring oxygen atom.

TABLE V INITIAL STEP 2 RESULTS % SRC INHIBITION IN CELLULAR MIMETICASSAY % Inhibition of 2 mM RR-src Inhibitor at Inhibitor Concentration () 42

47 (100 μM) 43

NON-ATPCOMPETITIVE Ortho: 39 (100 μM)Meta: 89 (100 μM)IC₅₀ = 18 μM,K_(i) = 10 μMPara: 23 (100 μM) 44

45 (100 μM) 45

NON-ATPCOMPETITIVE 51 (100 μM)IC₅₀ = 170 μM 46

Ortho: 42 (100 μM)Meta: In progressPara: 42 (100 μM) 47

40 (500 μM) 48

Ortho: 43 (100 μM)Meta: 30 (100 μM)Para: 45 (100 μM) 49

Ortho: 24 (100 μM)Meta: In progressPara: 54 (100 μM) 50

30 (100 μM)Lit. IC₅₀ = 118 nM Huang et al 51

37 (100 μM)Lit. IC₅₀ = 18 μM 52

41 (100 μM)Lit. IC₅₀ = 66 μMfor p56^(lck)

The inhibitor potencies in the src Cellular Mimetic assay can be furthercalibrated against other literature non-ATP, non-peptide src inhibitors.Two additional examples are 51 (ST 638, Shiraishi et al., 1989) which isa member of the “tyrphostin” family of erbstatin analogs (see Lawrence &Niu, 1998) and the natural product PTK inhibitor piceatannol 52 (Thakkaret al., 1993). In the Cellular Mimetic assay all of these knowninhibitors are less potent than had been reported suggesting that theassay is particularly demanding in terms of achieving high potency. Theinitial testing of src inhibitors is carried out using a singleconcentration (in triplicate) because commercial src is too expensive todo full IC₅₀ curves on every inhibitor. It should be mentioned, however,that an IC₅₀ dose response curve is not linear and the differencebetween ca. 50% inhibition at 100 μM and a ca. 90% inhibition at 100 μMis actually a factor of 10 and not a factor of 2 (e.g. 45 vs. 43-meta).Consequently, the literature src inhibitors 50–52 are greater than anorder-of-magnitude less active than the currently most potent inhibitor43-meta.

The discrepancies found within the literature reporting the potency ofthese inhibitors, the sensitivity to assay conditions described earlierwith the PKA inhibitors, and the lack of consistency among numerous labsand commercial protein kinase assay kits highlights this overlooked, butcrucial, problem in the field. Although the inhibitors produced by thepresent invention may be more potent under other assay conditions, theCellular Mimetic assay should be used, which mimics the intracellularphysical chemical conditions as closely as possible, as the primarypotency and rank order guide for evaluating the inhibitors beforechoosing compounds to proceed to whole cell or tissue assays. As will bediscussed in more detail later, the most potent naphthalene-basedinhibitor thus far from the Cellular Mimetic assay (i.e. 43-meta,IC₅₀=18 μM & K_(i)=10 μM) is also effective in specifically blockingpp60^(v-src) stimulated cell proliferation with a similar IC₅₀ of ca. 25μM. This suggests that not only is the Cellular Mimetic src assaypredictive, but also that this class of naphthalene-based inhibitors canreadily pass through cell membranes and inhibit intracellular src.

Analogs of a number of the naphthalene and indole inhibitors above canbe prepared with the boronic acid M₁ group in place of the M₁ OH and/orwith a S₂ hydrophobic specificity element attached for binding in thesrc P−1 site as illustrated in FIGS. 6 & 7. The naphthalene and indolescaffolds can then be taken through to Step 3 as described below. Eachtime Step 2 is repeated with new scaffolds from Step 1, the bestselectivity elements S₂ and/or S₃ which have discovered with previousscaffolds will be used in the combinatorial libraries of Step 3. Eventhough the optimal combination of M₁, S₂ an S₃ is likely to be differentfor each scaffold, those found optimal with the previous relatedscaffold (e.g. going from the naphthalene to the indole scaffold) shouldbe suitable for utilization as better initial specificity elements inStep 2 with the new scaffold. The same process will be repeated eachtime there is a need to try another scaffold until sufficient potency,selectivity and suitable pharmaceutical properties are achieved for thesrc inhibitors or, subsequently, for inhibitors of additionaltherapeutically important PTK's.

Some of the chemistry used to prepare the naphthalene inhibitors isdescribed in Marsilje 2000. For attaching a boronic acid functionalityin place of a M₁ hydroxyl groups in the src inhibitors from Table 5, thePd (0)-catalyzed cross-coupling methodology was used wherein either anaryl triflate (Ishiyama et al., 1997) or an aryl halide (Ishiyama, 1995)can be coupled with the commercially available pinacol ester of diboron.An illustrative example was have recently completed is given in FIG. 8.

The example shown in FIG. 8 demonstrates that it is possible toselectively triflate the less hindered OH at the M₁ position and haveproven this by its removal to 56 with subsquent ¹H NMR verification ofthe substitution pattern. The monotriflate 53 was then taken on to thedesired boronic acid 55 as indicated. The same reaction sequence alsoworks well for the regioisomer of 42 which corresponds to inhibitor 45from Table 5. The synthetic scheme shown in FIG. 9 can be followed, inorder to attach hydrophobic S₂ selectivity elements to the naphthalenescaffold.

The naphthalene chemistry can be converted to the solid phase inpreparation for synthesizing combinatorial libraries of this scaffold ina 96-well plate format. Thus far model chemistry has been carried out onthe less active naphthalene regioisomer represented by 44 because thiscompound is readily obtained from commercially available3,5-dihydroxy-2-naphthoic acid as describe in Marsilje 2000. Thesuccessful model reactions to date are shown in FIG. 10.

These model reactions demonstrate that it is possible couple thenaphthalene scaffold to the Wang resin (63) and then carry out chemistryon the triflate [in this case the Pd (0)-catalyzed cross-coupling to theboronic ester 64] followed by cleavage under mild conditions (65). Theester in 63 can also be saponified for subsequent coupling reactions toform amides containing the S₃ selectivity elements.

The naphthalene scaffold currently provides three diversity sites to beexplored in the combinatorial libraries, M₁, S₂ & S₃. Solid phasecombinatorial chemistry with 96-well plate reactors similar to that usedin previous studies (Pavia et al., 1996). The greatest number anddiversity of side chains will be used for S₃ followed by S₂ and then M₁for the reasons discussed earlier. One possible overall syntheticstrategy, based upon the synthetic model studies above, for preparingthese libraries is shown in FIG. 11.

Of course if problems arise with this route there are many otherpossibilities. For example, if the Mitsunobu coupling to give 67proceeds in too low a yield (due to the increased steric congestion ofthe added adjacent allyl group-but perhaps not a problem given the 92%loading obtained in FIG. 10) then the scaffold could be tethered to aresin through the carboxyl group, rather than the OH, using theacylsulfonamide “safety catch” linker (Backes et al., 1996) and form theamides last (the excess amines can be removed after cleavage byfiltering through an acidic resin). Likewise, other linkers and/orresins can be used if the reduction of the alkene in the presence ofbenzylic ethers (67 to 68) is desired but problematic. The first use ofthe chemistry proposed in FIG. 11 will be to simply prepare a library of96 amides, containing the boronic acid M₁ group, without having theallyl side chain in place so that these two potential complications willnot be a problem initially and the most promising S₃ elements can bequickly identified.

At least 14 S₂ hydrophobic side chains (includes linear, branched andcyclic) are identified for further study (28 if the correspondingalkenes are also explored) based upon the modeling of candidate sidechains into the P−1 site of the src model (FIG. 6) and on the commercialavailability of the needed halides to prepare the corresponding Wittigreagents. At least 96 commercially available amines are available whichwill provide potential S₃ specificity elements including: 1)hydrocarbons (4), 2) alkyl groups containing hydrogen bond acceptors(4), 3) alkyl groups containing both hydrogen bond acceptors and donors(19), 4) alkyl/aryl groups containing hydrogen bond acceptors and donors(25), 5) aryl hydrogen bond acceptors and donors (10), 6) heterocyclichydrogen bond acceptors and donors (20), 7) side chains containingcationic groups (4), 8) side chains containing anionic groups (9), andthe 3-amino phenol side chain from inhibitor 43-meta as an internalcontrol for src activity. A broad range of amines were included for S₃,in order not to overly bias the library here due to the higher level ofuncertainty for this binding site in the src model.

The indole scaffold can be developed into a combinatorial library inmuch the same fashion. In this case, the indole NH would be used as thetether point for attachement to the Wang (or other) resin since theanalogous Minsunobu reaction is known (Bhagwat & Gude, 1994). A largeamount of synthetic methodology has been developed for the synthesis ofsubsituted indoles and have designed a route to include the S₂hydrophobic side chain (see FIG. 7) (Ezquerra et al., 1996).

The triflate functionality formed in reaction 2 from intermediate 69(FIG. 11) can be converted to an amine (Wolfe et al., 1997) and then aseries of amides or other amine derivatives following the reactionsequence shown in FIG. 12. In fact, the triflate is a versatilesynthetic handle and could be converted into other functional groups aswell.

When the amine 72 is available, the known M₁'s (e.g. the sulfamic acidfrom src inhibitor 28 Table 5 and amide-acid 17 Table 3) can beevaluated with this more developed scaffold and evalulate some new aminederivatives as potential M₁'s. For example the hydrated tricarbonylamide M₁ group shown in structure 73 (and it's non-hydrated precursor)is accessible via the synthetic methodology (see Lai et al., 1996) andcould form a variety of interesting interactions with the conservedcatalytic residues.

Following the modeling procedure described above, a the series ofhydroxy-containing analogs of the boronic acid M₁ group shown in FIG. 13were modeled in the src and IRTK active sites and found the illustratedinteractions/binding modes as some of the interesting possibilities. Byphosphorylating the boronic acid additional interesting possibilitiesare available (e.g. suicide type inhibition via reaction of theresulting mixed anhydride with an active site nucleophile). The presenceof additional hydroxyl groups on the Tyr-mimetic phenyl ring isnecessary and common among many PTK inhibitors (e.g. Piceatannol 52,Table 5) and was shown to be benefical on the side chain with the PKAphosphonate inhibitors (e.g. 2 vs. 3 and 4, Table I). Consequently,adding one or more OH's to the boronic acid inhibitor M₁ design asillustrated in FIG. 13 may considerably enhance potency. These OH groupswould also extend the boronic acid side chain past the catalytic Asp andArg residues without suffering a penalty for covering them withhydrocarbon as was probably the case with the PKA boronic acid homologs(23 & 24, Table 3). One possible route to the hydroxyboronic acids 76 &77 utilizes the chiral boronic ester homologation methodology ofMatteson (e.g. see Matteson et al., 1987, 1988 & 1990).

In a preferred embodiment of the invention, the first module is producedby attaching the first module to a peptide scaffold. One or morefunctional groups are identified which preferentially bind to catalyticresidues of the protein kinase. Further, the first module is combinedwith the second module so that the second module substitutes for thepeptide scaffold.

Preferred first modules have a functional group such as boronic acid, ahydroxyl group, phosphonic acid, sulfamic acid, a guanidino group,carboxylic acid, an aldehyde, an amide, and hydroxymethylphosphonicacid. The compounds of the present invention may have two or morefunctional groups within the first module. More preferred modules areboronic acid groups, a hydroxyl group, or an amide group. An even morepreferred amide group is a vicinal tricarbonyl amide.

Preferred second modules include indole, naphthalene, biphenyl,isoquinoline, benzofuran, and benzothiophene. More preferred secondmodules are an indole or naphthalene. In some embodiments of theinvention more than one first module may be bound to the second module.In addition, the first module may have a linear chain comprising betweenone and three carbon atoms which links the first module to the secondmodule. In alternative embodiments, one of the carbon atoms in thelinear chain is substituted with a nitrogen, oxygen or sulfur atom.

The methods and compounds of the invention are broadly applicable to anyprotein kinase. Preferred protein kinases are protein tyrosine kinasesand protein serine kinases. Preferred protein tyrosine kinases arepp60^(C-Src), p56^(lck), ZAP kinase, platelet derived growth factorreceptor tyrosine kinase, Bcr-Abl, VEGF (vascular endothelial growthfactor) receptor tyrosine kinase, and epidermal growth factor receptortyrosine kinase, and epidermal growth factor receptor-like tyrosinekinases. A more preferred protein tyrosine kinase is pp60^(c-src).Preferred serine protein kinases include MAP (mitogen activated protein)kinase, protein kinase C, and CDK (cyclin dependent protein kinase).

The method of the present invention may further consist of adding one ormore specificity side chain elements to the combination of the first andsecond modules. Specificity side chains can increase potency andspecificity of the inhibitor.

Once a promising second module is identified it is not necessary torepeat all the steps of the method. Rather, the first module,specificity side chains, or a combination the two may be modified toimprove the original inhibitor, i.e an inhibitor which has an increasedability to inhibit protein kinase activity when compared to theunmodified first inhibitor.

The present method is designed to preferentially provide protein kinaseinhibitors which do not act by inhibiting ATP binding t the proteinkinase. Inhibitors of protein kinases may be potent but often lackspecificity and are therefore often not good drug candidates. Therefore,protein kinase inhibitors which inhibit protein kinase activity but doesnot inhibit or only weakly inhibit ATP binding to the protein kinase arepreferred.

The present invention also provides a method for testing compounds foran ability to inhibit protein kinase activity. Compounds are produced asdescribed above. The activity of the protein kinase is measured in thepresence of the inhibitor at the same temperature, pH, ionic strength,osmolarity, and free magnesium concentration as found in a cell whichexpresses the protein kinase. The level of protein kinase activity iscompared to the level of activity from the protein kinase without thepresence of the inhibitor. Such an assay system which mimicsphysiological conditions provides the most relevant inhibition data. Theassay may be conducted in an automated assay system. Furthermore, theassay may be combined with a combinatorial chemistry method to rapidlyscreen numerous candidates.

The Pierce 96-well plate non-radioactive ELISA PTK assay method may beadapted to the Cellular Mimetic assay conditions for initial srcscreening of the 96-well plate combinatorial libraries. This highthroughput assay utilizes the same RR-SRC peptide substrate, except thatit is biotinylated so that it can be attached to the NeutrAvidin-coatedwells in their commercial 96-well plates. This high throughputinhibition assay can be run by incubating src with the RR-SRC substrateprebound to the wells followed by adding their anti-phosphotyrosineantibody (PY20)-horseradish peroxidase (HRP) conjugate and their HRPsubstrate to quantitate the level of phospho-RR-SRC produced viameasuring the level of HRP product with a 96-well plate UV reader.Standard low throughput P³²-ATP radioactive assays have been used, but a96-well plate format is preferred, especially with a non-radioactiveassay if possible. As very potent src inhibitors are developed, a panelof protein kinase assays could be set up with up to ca. 6 commerciallyavailable protein kinases (mostly PTKs), using the Cellular Mimeticprotein kinase assay conditions, and test these inhibitors across thepanel to obtain an initial assessement of specificity. A more completespecificity assessment, involving the full ca. 2,000 protein kinases,will need to be conducted in cell culture and in vivo through additionalcollaborations at the appropriate time.

Active src inhibitors can be studied in a set of side-by-side cell-basedassays using normal rat kidney (NRK) cells and a temperature-sensitivepp60^(v-src) tranformant of this cell line (LA25). The LA25 transformantengages in anchorage- and serum-independent growth at the “permissive”temperature of 33° C. due to activation of pp60^(v-src) but not at the“non-permissive” temperature of 40° C. at which pp60^(v-src) is notactivated (Li et al., 1996). The use of this pair of closely relatedcell lines for testing the src inhibitors at both the permissive andnon-permissive temperatures allows one to determine if a given srcinhibitor is blocking cell growth due to specific blockade of the srcsignalling pathway, by a different mechanism or by a general cytotoxiceffect. Results from initial testing of the non-peptide src inhibitor43-meta (Table V) in this pair of cell lines are shown in FIG. 14.

As shown in this graph the growth of the LA25 cells at the permissivetemperature of 33° C. is inhibited by ca. 50% at a 25 μM concentrationof 43-meta relative to the LA25 cell growth at the non-permissive 40° C.as a control. The lack of cell toxicity of 43-meta is evidenced by thefact that as its concentration is increased up to 400 μM, the baselgrowth of the NRK non-transformed cells, the LA25 cells at thenon-permissive 40° C. and the LA20 cells at the permissive temperatureof 33° C. (but with pp60^(v-src) fully inhibited by 43-meta) not onlydoes not decrease but actually increases somewhat (presumably due to anon-src related activity of this compound). Since the 43-meta solutionswere prepared with a low concentration of DMSO for solubilization a DMSOcontrol was also run at the same concentration. A more completedose/response curve centered around 25 μM will be run.

In another embodiment, the present invention provides a method ofinhibiting a protein kinase. The protein kinase is contacted with acompound having a first module which has a functionality for binding tocatalytic residues of the protein kinase and a second module whichprovides a non-peptide scaffold. The combination of the first and secondmodules inhibits the protein kinase activity.

A preferred non-peptide protein tyrosine kinase inhibitor provided bythe present invention has the following formula:

wherein R1 is H or OH, R2 is H or OH, R3 is OH or H, and R4 is CH₂,CH(CH₃)R, or CH(CH₃)S, R5 is OCH₃, H, or OH, R6 is OCH₃, F, H, or OH,and R7 is OCH₃, H, OH, CO₂H, CO₂CH₃, CH₂CO₂H, or CH₂CO₂CH₃. In a morepreferred embodiment, the non-peptide protein tyrosine kinase inhibitorinhibits the activity of pp60^(c-src) tyrosine kinase.

Another preferred non-peptide protein tyrosine kinase inhibitor has thefollowing formula:

where R1 is OH or H, R2 is OH or H, R3 is OH or H, R4 is OH or H, R5 isOH, OMe, or H, R6 is OH, OMe, or H, R7 is OH, OMe, or H, and X is 0or 1. In a more preferred embodiment, the non-peptide protein tyrosinekinase inhibitor has the above structure and R1 is OH, R2 is OH, R3 isH, R4 is H, R5 is OMe, R6 is H, R7 is H, and X is 1.

Yet another preferred non-peptide protein tyrosine kinase has theformula:

The present invention further provides a method of treating a condition,responsive to a protein kinase inhibitor, in a patient. An effectivedose of a protein kinase inhibitor is administered to a patient. Theprotein kinase inhibitor has a first module having a functionality forbinding to catalytic residues of the protein kinase and a second modulewhich provides a non-peptide scaffold, where the combination of thefirst and second modules inhibits protein kinase activity.

Finally, promising src inhibitors can be screened in primary human tumortissue assays, particularly to look for synergy with other knownanti-cancer drugs.

EXAMPLES Example 1 Design, Synthesis and Activity of Non-Atp CompetitiveHydroxynaphthalene Derivative Inhibitors of pp60^(c-Src) Tyrosine Kinase

The crystal structure of the autoinhibited human IRTK catalytic domain(Hubbard et al., 1994) was used to carry out qualitative molecularmodeling studies (SYBYL™, 6.4, Tripos Inc., St. Louis) wherein anaphthalene ring was superimposed upon the IRTK Tyr 1,162. The IRTKregion containing Tyr 1,162 folds back into the active site, with Tyr1,162 positioned analogous to a phosphorylatable Tyr in a peptidesubstrate, thereby autoinhibiting the tyrosine kinase. Thissuperimposition indicated that an amide carbonyl should be placed at the2-position (Scheme 1) of the

naphthalene ring to mimic the Tyr 1,162 carbonyl and a hydroxyl groupshould be positioned at the 6-position to mimic the Tyr 1,162 hydroxylgroup. These modeling studies also indicated that a hydroxyl group atthe 3-position could mimic the Tyr 1,162 NH.

In order to test these design concepts experimentally, the 2-positioncarbonyl group was appended as either a methyl ester or as a series ofamides (Table 6). The hydroxy N-phenyl (X=0) and N-benzyl (X=1) amideswere chosen based upon the increase in pp60^(c-scr) inhibitor potencyobserved with iminochromene analogs containing appended hydroxy N-phenylamide side-chains (Huang et al., 1995). Analogs wherein the 6-hydroxylgroup was either deleted or moved were also prepared to determine if adrop in potency occurs as predicted from the modeling studies.

The series of 2-carbonyl-3,5-dihydroxy naphthalene inhibitors (1a,2a–2d, 2i–2l, 2o–2p) and 2-carbonyl-3,7-dihydroxy naphthalene inhibitors(1c, 2t–2u) were synthesized from commercially available (Aldrich)3,5-dihydroxy-2-naphthoic acid and 3,7-dihydroxy-2-naphthoic acid,respectively. The methyl esters 1a and 1c were obtained by refluxing therespective acid starting materials for 48 h in methanol pre-saturatedwith HCl gas. The amides (2a–2d, 2i–2l, 2o–2p, 2t–2u) were synthesizedby coupling the respective carboxylic acid with commercially available(Aldrich or Lancaster) amines using one of two methods. The first methodutilized the NBS/PPh₃ methodology as described by Froyen (Froyen, 1997).The second method utilized IIDQ (Aldrich) as the coupling reagent. Thecarboxylic acid was first reacted with 1.0 eq. IIDQ in anhydrous DMF atroom temperature for 24 h. The respective amine (2.0 eq.) was then addedneat and the reaction was heated to 80° C. for 2–6 hours. After aqueousworkup, purification was achieved by silica gel chromatography andprecipitation from CH₂Cl₂/hexane, followed by preparative C-18 RP-HPLC(CH₃CN/H₂O), if necessary. The benzyl amines were commercially availableonly as their corresponding hydroxyl protected methyl ethers.Consequently, after amide formation, the hydroxyl groups weredeprotected by treatment with 6 eq. BBr₃ in DCM for 1 minute at −78° C.followed by 1 hour at room temperature.

TABLE 6 pp60^(c-src) inhibitory activity of hydroxynaphthalenederivatives and select

published inhibitors.^(a,b,c) % Inhibition at 100 μM (std. Compd R1 R2R3 R4 R5 R6 R7 X dev.) IC50 (μM) 1a OH OH H H N/A N/A N/A N/A  5 (+/−2)n.t. 1b OH H OH H N/A N/A N/A N/A 47 (+/−3) n.t. 1c OH H H OH N/A N/AN/A N/A 19 (+/−6) n.t. 1d NH₂ H H H N/A N/A N/A N/A inactive n.t. 2a OHOH H H OH H H 0 12 (+/−4) n.t. 2b OH OH H H H OH H 0 51 (+/−1) 150 2c OHOH H H H H OH 0 60 (+/−7) n.t. 2d OH OH H H OH H OH 0 14 (+/−2) n.t. 2eOH H OH H OH H H 0 39 (+/−5) n.t. 2f OH H OH H H OH H 0 89 (+/−1)  16 2gOH H OH H H H OH 0 23 (+/−5) n.t. 2h OH H OH H OH H OH 0 56 (+/−1) n.t.2i OH OH H H H OMe H 0 33 (+/−5) n.t. 2j OH OH H H H H OMe 0 35 (+/−8)n.t. 2k OH OH H H OMe H H 1 13 (+/−3) n.t. 2l OH OH H H H H OMe 1 14(+/−2) n.t. 2m OH H OH H OMe H H 1 inactive n.t. 2n OH H OH H H H OMe 1 4 (+/−7) n.t. 2o OH OH H H OH H H 1 41 (+/−2) n.t. 2p OH OH H H H H OH1 49 (+/−4) n.t. 2q OH H OH H OH H H 1 42 (+/−2) n.t. 2r OH H OH H H OHH 1 55 (+/−3) n.t. 2s OH H OH H H H OH 1 42 (+/−3) n.t. 2t OH H H OH HOH H 0 68 (+/−5) n.t. 2u OH H H OH H OH H 1 40 (+/−3) n.t. 2v H H OH H HOH H 0 45 (+/−5) n.t. Iminochromene 9TA  30 (+/−15) Lit⁸: 0.118Piceatannol 41 (+/−2) Lit¹³: 66 (lck) ST-638 37 (+/−5) Lit¹⁴: 18Emodin^(d) 22 (+/−3) Lit¹⁵: 38 Tyrophostin A47 43 (+/−3) Table 6Footnotes: ^(a)The previously described assay procedure (Lai et al.,1998) was used with the following assay components, final concentrationsand conditions: 50.0 mM MOPS, 4.02 mM MgCl₂, 6.00 mM K₃ citrate (used asa Mg²⁺ buffer to stabilize the free Mg²⁺ at 0.5 mM), 99.0 mM KCl, 10.0mM 2-mercaptoethanol, 198 μM ATP, 19.8 μM ADP, 10 U full length humanpurified recombinant pp60^(c-src) (Upstate Biotechnology Inc.), 2.00 mMRR-SRC, 4.0% DMSO, pH 7.2, 37° C. These overall assay conditions havebeen shown (Choi, 1999) to reproduce the intracellular conditions of pH,temp., free Mg²⁺ (0.5 mM), ionic strength, osmolality, ATP/ADP andreduction potential. ^(b)All new compounds were characterized by protonNMR, EI or FAB(+) MS and are pure by TLC. ^(c)N/A = Not applicable, n.t.= Not tested. ^(d)ATP-competitive.

The series of 2-carbonyl, 3,6-dihydroxy naphthalene inhibitors (1b,2e–2h, 2 m–2n, 2q–2s) were synthesized from 3,6-dihydroxy-2-naphthoicacid 6 using the methods described above. The synthesis of intermediate6 that was developed is shown in Scheme 2 beginning with commerciallyavailable 2,7-dihydroxynaphthalene 3 (Aldrich).

Compound 1d was synthesized from 3-amino-2-naphthoic acid (Aldrich) byreaction with TMS-diazomethane in DCM at room temperature. Compound 2vwas synthesized from 6-hydroxy-2-naphthoic acid (Aldrich) using theamidation method described by Froyen (Froyen, 1997).

Kinase assay conditions have been shown to influence the measuredinhibitory activity (Lawrence et al., 1998). Consequently, in order toaccurately determine the relative potency of the newly designed class ofpp60^(c-src) inhibitors, the inhibitory activity of four previouslypublished, non-ATP competitive PTK inhibitors, was also tested.Piceatannol, ST-638, and Tyrphostin A47 were chosen because they arecommercially available (Sigma or Calbiochem), and are representative ofthe spectrum of known non-ATP competitive PTK inhibitors. Emodin(Calbiochem) is ATP-competitive when analyzed with the tyrosine kinasep56^(lck). Previously, iminochromene 9TA was the most potent non-ATPcompetitive pp60^(c-src) inhibitor reported (Huang et al., 1995). Sinceiminochromene 9TA was not commercially available, it was synthesizedusing a novel route by converting 3-Aminophenol to the correspondingTBDMS ether (1.1 eq. TBDMS-Cl, 1.1 eq. DIEA, 5 mol % DMAP, DMF, 24 h,rt, 71%). The resulting aniline was coupled using 2.0 eq. of cyanoaceticacid (1.1 eq. EDCI, 1.1 eq. TEA, DMF, 18 h, 75° C., 70%). Condensationof the resulting amide with 1.2 eq. of 2,3-dihydroxybenzaldehyde (cat.piperidine, abs. EtOH, 2 h, 60° C.) followed by deprotection (1.1 eq.TBAF, THF, 15 m, 43% overall) gave iminochromene 9TA with satisfactoryelemental, FAB(+)MS and ¹H NMR analysis after purification by flashchromatography (10:1, DCM:MeOH).

The inhibitory activities shown in Table 6 for compounds 1a–d and 2a–2vwere determined using purified, full length, human recombinantpp60^(c-src). Due to the number of compounds tested, and the associatedcost, their rank order potencies were first determined at a constantinhibitor concentration (100 μM). As predicted by the modeling studies,based upon analogy to the IRTK Tyr 1,162 hydroxyl group, a preferencefor positioning the naphthalene hydroxyl group on carbon 6 vs. 5 or 7was observed in both the ester (1b, 47% vs. 1a, 5% & 1c, 19%) and amide(e.g. 2f, 89% vs. 2b, 51% & 2t, 68%) series. The prediction thatattaching a hydroxyl group at naphthalene carbon 3 (mimicking the Tyr1,162 NH) would improve potency was also confirmed (2f, 89% vs. 2v,45%). Finally, the prediction that extending the inhibitor as an amideat the 2 position (mimicking the peptide bond) could further improvepotency was confirmed as well (e.g. 2f, 89% vs. 1b, 47%).

The data provided in Table 6 demonstrate that moving the hydroxyl groupfrom the optimal 6 position to the adjacent naphthalene carbon 5 resultsin a different structure activity profile with regard to the optimalconcurrent positioning of the hydroxyl group(s) in the amide side chain(e.g. 2f/2g vs. 2b/2c). Also of note is the replacement of the amideside chain hydroxyl group with a corresponding methoxy group incompounds 2i–2n. In the case of the N-phenyl amides (2i–2j), theiractivity, relative to the corresponding hydroxy amides (2b–2c), was notreduced as significantly as in the case of the N-benzyl amides (2k–2nvs. 2o–2q, 2s). This suggests that in the benzyl derivatives, the amideside chain hydroxyl groups either interact with the enzyme as hydrogenbond donors, or the methoxy groups are too large to fit in the bindingsite.

A more quantitative analysis of the selectivity for positioning ahydroxyl group on carbon 6 vs. 5 is provided by comparing the IC₅₀'s of2f (16 μM) vs. 2b (150 μM), respectively. These results also confirmthat a drop in % inhibition from ca. 90% to ca. 50% represents an orderof magnitude difference in potency, as expected. Similarly, a drop in %inhibition from ca. 50% to 10% would represent another order ofmagnitude difference in potency.

A direct comparison of the most potent inhibitor from this series,compound 2f, with the five previously reported PTK inhibitors shown inTable 6 demonstrates that, under these assay conditions, 2f is morepotent by one to two orders of magnitude. Interestingly, iminochromene9TA was previously reported (Huang et al., 1995) to have an IC₅₀ of 118nM against pp60^(c-src), and was the most potent known non-ATPcompetitive pp60^(c-src) inhibitor, but under the current assayconditions only a 30% inhibition at 100 μM was observed. These resultsre-emphasize (Lawrence et al., 1998) the importance of comparing proteinkinase inhibitors under identical assay conditions.

A goal of these studies was to obtain non-peptide pp60^(c-src)inhibitors which do not compete with ATP. Consequently the % inhibitionof pp60^(c-src) by 2f and 2b at constant inhibitor concentrations wasmonitored as a function of increasing [ATP] up to a cellular mimetic 1mM level. Since the [ATP] had little effect on the % inhibition, both 2fand 2b are non-competitive inhibitors with respect to ATP. The %inhibition was measured using ATP concentrations of 200, 500 & 1,000 μMwhile holding the inhibitor concentration constant. If the inhibitor isdirectly competing with ATP, then this 5-fold overall increase in [ATP]is equivalent to decreasing the inhibitor concentration 5-fold in termsof the effect on % inhibition. Consequently the % inhibition shoulddecrease to the value observed in the IC₅₀ dose-response curve (obtainedwith 200 μM ATP) for ⅕ of the set inhibitor concentration used in thisexperiment if direct competition with ATP is occurring. For inhibitor 2f(set at 25 μM) a 62% (+/−5), 54% (+/−3) and 50% (+/−1) inhibition at 200μM, 500 μM and 1,000 μM ATP, respectively, was obtained whereas thelevel of inhibition should have dropped to ca. 20% at 1,000 μM ATP ifdirect competition with ATP were occurring. Similarly, for inhibitor 2b(set at 300 μM) an 84% (+/−1), 81% (+/−1) and 77% (+/−2) inhibition at200 μM, 500 μM and 1,000 μM ATP, respectively, was obtained. The highcost of many kinases has stimulated other researchers to monitorinhibitor potency as a function of increasing [ATP] for the same purpose(Saperstein et al., 1989; Burke et al., 1993; Davis et al., 1989; Daviset al., 1992; Faltynek et al., 1995; and Sawutz et al., 1996).

In summary, structure-based design has produced a series ofhydroxynaphthalene pp60^(c-src) non-peptide inhibitors that do notcompete with ATP. Results with compounds from this series in cell-basedassays, as well as detailed kinetic studies under various assayconditions, will be reported in due course. An extension of these designconcepts from the naphthalene scaffold to an indole scaffold is reportedin the following paper.

Example 2 Design, Synthesis and Activity of Non-ATP CompetitiveHydroxyindole Derivative Inhibitors of pp60^(c-Src) Tyrosine Kinase

In the preceding example, the structure-based design of a series ofpp60^(c-src) inhibitors utilizing a naphthalene scaffold is described.These compounds were designed to bind in the peptide substrate sitebecause of the potential for greater selectivity and efficacy in acellular environment relative to the alternative ATP

substrate site. This example presents an extension of these designconcepts to a series of pp60^(c-src) inhibitors based upon an indolescaffold. Once again the crystal structure of the autoinhibited insulinreceptor PTK (IRTK) was used to carry out qualitative molecular modelingstudies, except in this case an indole ring was superimposed upon theIRTK Tyr 1,162. This superimposition indicated that the indole NH canmimic the Tyr 1,162 NH, that a carbonyl should be placed at the2-position, and a hydroxyl group at the 5 position to mimic the Tyr1,162 carbonyl and OH, respectively (Scheme 1). The conceptualcyclization of Tyr 1,162 to the smaller 5-membered ring of an indoleillustrated in Scheme 1, relative to a 6-membered ring in the case ofthe naphthalene scaffold (Karni et al., 1999), results in a movement ofthe optimal positioning of the OH from carbon 6 in the naphthalenescaffold to carbon 5 in the indole scaffold.

The indole amide derivatives containing hydroxy phenyl/benzyl sidechains 2d–f, 2j–l (Table 7), respectively, were selected based upon theincrease in pp60^(c-src) inhibitor potency observed for the analogousnaphthalene-based hydroxy phenyl amides reported in the previousexample. The corresponding methyl ethers 2a–c,g–i,v are precursors inthe synthesis. The additional analogs shown in Table 7 were prepared tobegin expanding the range of side chains beyond the hydroxy/methoxygroups that have now been extensively probed with both the indole andnaphthalene scaffolds.

The indole amides containing only hydroxy or methoxy side chains weresynthesized as illustrated:

The 2-indolecarboxylic acid derivative, the methoxyphenyl amine (1.1 eq,Aldrich, Lancaster or Fluka), and the coupling reagent PyBOP(benzotriazol-1-yloxy)tripyrrolidino-phosphonium-hexafluorophosphate) (1eq, Fluka) were dissolved in anhydrous DMF. The solution was cooled to0° C. under argon and then diisopropylethylamine (DIEA, 3 eq) was added.The reaction was stirred at 0° C. for 1 m followed by 1 hour at roomtemperature. After workup the residue was purified by silica gelchromatography.

The methyl ethers were cleaved with boron tribromide (1 M in DCM,Aldrich) when desired. The indole amide methyl ether was suspended indry DCM and cooled to −78° C. under argon. One equivalent of BBr₃ wasadded for each heteroatom in the starting material plus one excessequivalent. The resulting dark red solution was stirred at −78° for 30 mand then at room temperature for 1–2 hours. The reaction was quenchedwith water (10 minutes) before workup.

TABLE 7 pp60^(c-src) inhibitory activity of hydroxyindolederivatives.^(a,b,c)

% Inhibition at Compd R1 R2 R3 R4 R5 R6 R7 100 μM (std. dev.) 1a H OH HCH₃ N/A N/A N/A 40 (+/−5) [at 500 μM] 1b H OH OH CH₂CH₃ N/A N/A N/A 28(+/−3) 2a H OH H — OCH₃ H H  3 (+/−1) 2b H OH H — H OCH₃ H 21 (+/−2) 2cH OH H — H H OCH₃ 39 (+/−9) 2d H OH H — OH H H 43 (+/−1) 2e H OH H — HOH H 30 (+/−6) 2f H OH H — H H OH 45 (+/−3) 2g H OH H CH₂ OCH₃ H H 21(+/−5) 2h H OH H CH₂ H OCH₃ H  7 (+/−6) 2i H OH H CH₂ H H OCH₃ 18 (+/−4)2j H OH H CH₂ OH H H 24 (+/−3) 2k H OH H CH₂ H OH H 74 (+/−2) [IC₅₀ = 38μM] 2l H OH H CH₂ H H OH 54 (+/−2) 2m H OH H CH₂CH₂ H H OH 21 (+/−7) 2nH OH H CH₂ H H CO₂H not active 2o H OH H CH₂ H H CO₂CH₃ 11 (+/−4) 2p HOH H — H H CH₂CO₂H  7 (+/−6) 2q H OH H — H H CH₂CO₂CH₃ 32 (+/−7) 2r H OHH — H F H 21 (+/−7) 2s H OH H CH₂ H F H 57 (+/−6) 2t H OH OH CH₂ H OH H26 (+/−2) 2u H H OH CH₂ H OH H 56 (+/−6) 2v H H H CH₂ H H OCH₃  4 (+/−4)2w H H H CH₂ H H OH 36 (+/−4) 2x OH H H CH₂ H OH H 60 (+/−3) 2y H OH HCH(CH₃)R H OH H 15 (+/−3) 2z H OH H CH(CH₃)S H OH H 13 (+/−7) ^(a)Allcompounds were tested as described in the preceding paper.⁵ ^(b)Allcompounds were characterized by proton NMR, FAB(+) MS and are pure byTLC. ^(c)N/A = Not applicable.

Using this synthetic route, the series of 5-hydroxyindole amideinhibitors 2a–m,y,z were prepared from 5-hydroxy-2-indolecarboxylicacid. The 4- and 6-hydroxyindole amides (2x,u, respectively) weresynthesized from methyl 4-methoxy-2-indolecarboxylate and methyl6-methoxy-2-indolecarboxylate, respectively. The 5,6-dihydroxyindoleamide 2t was prepared from ethyl 5,6-dimethoxyindole-2-carboxylate.Sonication of the esters in 1 N NaOH for 1 h provided the correspondingcarboxylic acids for coupling. The des-hydroxy indole amides 2v,w weresynthesized from indole-2-carboxylic acid. All of the indole startingmaterials were commercially available (Aldrich or Lancaster).

The fluoro inhibitors 2r,s were likewise prepared from the correspondingfluorophenyl amines (Aldrich). The inhibitors containing esters orcarboxylic acids on the amide side chain, 2n–q, were prepared from thecorresponding amino carboxylic acids (Aldrich). The side chaincarboxylic acid was first protected as a methyl ester (anh. MeOHpre-saturated with HCl, reflux, 1d), followed by PyBOP coupling (asabove), then saponification back to the carboxylic acid when desired.

The methyl ester 1a was prepared by refluxing a solution of thecarboxylic acid overnight in anhydrous methanol pre-saturated with HClgas. The ethyl ester 1b was prepared by BBr₃ deprotection of ethyl5,6-dimethoxyindole-2-carboxylate as above. All of the inhibitors listedin Table 7 were purified by silica gel chromatography.

As in Marsilje 2000, the rank order activity of this series ofpp60^(c-src) inhibitors was first determined at a constant inhibitorconcentration (Table 7). The same inhibitor concentration (100 μM) wasused for the current indole series of inhibitors, the previousnaphthalene series of inhibitors, and five non-ATP competitiveliterature PTK inhibitors (see preceding paper). This allowed anefficient rank order comparison of 59 compounds in total under identicalassay conditions.

The modeling studies predicted that a hydroxy group at carbon 5 of theindole scaffold would be optimal. Comparison of the 5-hydroxy indoleinhibitor 2k (74%) with the analogous 6-hydroxy indole inhibitor 2u(56%) and 4-hydroxy indole inhibitor 2x (60%) confirms this prediction,although the preference is not strong. The prediction that a hydroxygroup at carbon 5 will improve the activity (relative to no hydroxygroup) is confirmed by comparing the 5-hydroxy indole inhibitor 21 (54%)with the corresponding des-hydroxy inhibitor 2w (36%).

Extending the indole inhibitors as aryl amides at carbon 2 improvedpotency, as expected based upon the previous naphthalene inhibitors. Forexample, the meta-hydroxybenzyl amide indole 2k gives 74% inhibition at100 μM whereas the analogous methyl ester 1a gives only 40% inhibitionat 500 μM. Interestingly, comparing the 5,6-dihydroxy ethyl ester 1b(28%) to the corresponding aryl amide 2t (26%) shows that thesimultaneous presence of the second hydroxy at carbon 6 prevents thepotency enhancement normally provided by the otherwise preferredmeta-hydroxybenzyl amide side chain. This amide side chain was the bestof the current series when the 5-hydroxyl group is present alone (2k,74%) and still gave good inhibition when a 6-hydroxy group was presentalone (2u, 56%). Also, the simultaneous presence of two hydroxy groupsat carbons 5 & 6 seems well tolerated in the absence of an amide sidechain (1b vs. 1a & 2e). This data suggests that a change in the bindingorientation of the indole scaffold may have occurred due to the presenceof the second hydroxy group and that a different amide side chain maynow be preferred. The optimal combination of side chains at carbons 4–7(including functional group replacements for hydroxy groups (Lai et al.,1999)) and amide side chains is currently under investigation.

In general, the indole scaffold structure-activity-relationships (“SARs”) revealed by the data in Table 7 parallels that reported in thepreceding paper for the naphthalene scaffold. In both cases positioninga hydroxy group on the scaffold analogous to the Tyr 1,162 OH, asidentified by modeling studies, provided the highest potency. Movingthis hydroxy group to one of the adjacent carbons reduced the potency,but not dramatically, in both cases. Extending both scaffolds with arylamides at the position identified by the modeling studies to mimic theTyr 1,162 peptide bond improved the potency. With both scaffolds,substitution of a methoxy group for the hydroxy groups on the amide sidechain usually reduced potency, and did so to a greater extent with thelonger benzylamide side chain (e.g. 2k, 74% vs. 2h, 7% compared to 2e,30% vs. 2b, 21%). The major difference in the SARs for these twoscaffolds is that the 5-hydroxyindole scaffold prefers the longerm-hydroxybenzyl amide side chain (2k, 74% vs. 2e, 30%) whereas theanalogous 3,6-dihydroxynaphthalene scaffold prefers the shorter amideside chain derived from m-hydroxyaniline. The 5-hydroxyindole scaffoldshowed essentially no preference for the position of the hydroxyl groupon the shorter amide side chain (2d–f) whereas with the longerhydroxybenzyl amide side chain a significant preference for the metaposition was observed (2j–l). In the case of the3,6-dihydroxynaphthalene scaffold the opposite was observed.

Additional molecular modeling studies were carried out to further probethe preference for a longer amide side chain with the indole scaffold.The most active naphthalene inhibitor 3 from the previous report wasused as a template upon which the analogous indole inhibitor 2e and thehomologated indole inhibitor 2k were superimposed. The three mostimportant side chain functional groups in naphthalene inhibitor 3 areconsidered to be the 6-hydroxy group (H-bond donor and acceptor), the

hydrogen from the 3-hydroxy group (H-bond donor), and the side chainhydroxy group (H-bond acceptor) based upon the rational design and SARfor both series of inhibitors. This three point pharmacophore model isidentified in both series by asterisks in Scheme 3.

The “multifit”, energy minimization and “fit atoms” facilities withinSYBYL™ (6.4, Tripos, St. Louis) were used in sequence to superimpose 2eand 2k onto 3. This overall fitting process was carried out with springconstants (multifit) and weights (fit atoms) chosen such that thehighest emphasis was on optimally superimposing the scaffoldpharmacophore O's and H's (100), followed by the side chain O's (10) andthen the intervening amide bond (1). The “multifit” process adjusted theconformations for maximum pharmacophore fit, the subsequent minimizationproduced the nearest local minimum energy conformations and finally the“fit atoms” process produced the best pharmacophore superimposition ofthese minimized conformations. As expected, the scaffold pharmacophoreO's and H's of both 2e and 2k superimposed closely and similarly uponthe corresponding atoms in 3 (all within ca. 0.50 A^(o)). However, theside chain pharmacophore O's of 2e and 2k differed significantly intheir superimposition on the corresponding O of 3, with displacements of1.8 A^(o) vs. only 0.08 A^(o) respectively. This close fit of the threekey pharmacophore sites between 2k and 3 provides a rationalization fortheir potency differing by only a factor of 2.4 (IC₅₀'s 38 μM vs. 16 μM,respectively).

Extending the amide side chain by another carbon atom reduced theactivity (2m, 21% vs. 21, 54%). Adding a methyl group to the benzyliccarbon of 2k, in either stereochemistry, greatly reduced the activity(2y, 15% & 2z, 13% vs. 2k, 74%). Replacing the side chain hydroxy group(in the para position) with a carboxylate anion (2n, 0% vs. 21, 54% and2p, 7% vs. 2f, 45%) reduced the activity whereas the correspondingmethyl esters (2o, 11% & 2q, 32%, respectively) showed a smaller loss ofpotency. Importantly, replacing the side chain hydroxy group with afluorine maintained much of the potency (2s, 57% vs. 2k, 74% and 2r, 21%vs. 2e, 30%). Consequently, the fluoro analog 2s has only one hydroxygroup left for potential Phase II metabolism (e.g. glucuronideformation), and this remaining hydroxy group is a current target forreplacement (Lai et al., 1998).

Using the same method as in the preceding example (Marsilje 2000), themost potent inhibitor from the current indole series (2k) was analyzedfor ATP competition by monitoring the % inhibition at increasing [ATP]while holding the inhibitor concentration constant. Since the [ATP] hadlittle effect on the % inhibition (The % inhibition was 46% and 41% with2k at 45 μM and [ATP] at 200 μM or 1,000 μM, respectively.), 2k isnon-competitive with respect to ATP under these assay conditions.

In summary, an indole scaffold has been designed, and an initial SARcarried out, for the development of non-ATP competitive pp60^(c-src)inhibitors. The potency of the best indole-based inhibitor from thecurrent series was found to be close to that of the bestnaphthalene-based inhibitor. The % inhibition was 46% and 41% with 2k at45 μM and [ATP] at 200 μM or 1,000 μM, respectively.

Example 3 Synthesis of Indole Derivative Protein Kinase Inhibitors

The following results show the synthesis and testing of indole derivedprotein kinase inhibitors. Four reaction schemes are provided andseparately followed by experimental details for the preparation of thefinal product of each of these reaction schemes. These final productsare examples of indole-base tyrosine kinase inhibitors wherein thesynthesis with preferred R groups is illustrated (boronic acid, Scheme1; OH, Scheme 2; an aliphatic amide extension, Scheme 3; and aphosphonic acid Scheme 4).

Methyl5-hydroxy-2-indolecarboxylate (1)

Dissolved 3.50 g 5-hydroxy-2-indolecarboxylic acid in anh. MeOHpresaturated with HCl gas. Refluxed for 48 hours. Concentrated in vacuoand triturated with AcCN x3 to remove residual acid. Filtered throughsilica plug with EtOAc to remove baseline contamination. Recovered 4.32g (quant. yield) TLC R_(f)=0.78 (EtOAc) ¹H NMR (DMSO-d₆): 3.82 (s, 3H),6.78 (d, J=8.8 Hz, 1H), 6.88 (s, 1H), 6.93 (s, 1H), 7.23 (d, J=8.8 Hz,1H), 8.90 (s, 1H) 11.62 (s, 1H) FAB(+) MS m/e 191.9 (M+1)

Methyl 5-[(trifluoromethyl)sulfonyloxyl]indole-2-carboxylate (2)

Added 150 ml anh. DCM to 3.24 g (17 mmol) methyl5-hydroxy-2-indolecarboxylate (1) and 6.67 g (18.7 mm) n-phenyltrifluoromethane sulfonamide at 0° C. Added 2.6 ml triethylaminedropwise at which point clear yellow solution formed. Stirred at 0° C.for 1 hour. Warmed to room temperature and stirred for 2 hours.Concentrated in vacuo and purified through silica gel column (1/1EtOAc/hexanes). Recovered 4.69 g (86%). TLC R_(f)=0.63 (1/1EtOAc/hexanes). HPLC R_(f)=20.879 1H NMR (DMSO-d₆): 3.87 (s, 3H), 7.25(s, 1H), 7.31 (d, J=9.2 Hz, 1H), 7.55 (d, J=9.2 Hz, 1H), 7.80 (s, 1H),12.34 s, 1H) FAB(+) MS m/e 323.1 (M+1).

Methyl 5-methylindole-2-carboxylate,4,4,5,5-tetramethyl-1,3,2-dioxaborolanemethyl (3)

500 mg 1.55 mmol methyl5-[(trifluoromethyl)sulfonyloxy]indole-2-carboxylate (2), 37.9 mg (0.05mmol) PdCl₂ (dppf), 432 mg (1.7 mmol) bispinacolatodiboron, 454.8 mg(4.65 mmol) potassium acetate, and 25.7 mg (0.05 mmol) dppf were addedto a flask and vacuum dried at 40° C. for 2 hours. Added 20 ml anhdioxane and heated to 80° C. overnight. Reaction turned black as Pdblack precipitated out. Filtered off catalyst and ran silica plug toremove baseline impurities. TLC R_(f)=0.51 (1/4 EtOAc/Hexane) Crudeproduct was taken through to next reaction.

Methyl 5-boronyl indole-2-carboxylate (4)

391.2 mg (1.3 mmol) methyl 5-methylindole-2-carboxylate,4,4,5,5-tetramethyl-1,3,2-dioxaborolanemethyl (3) was dissolved inEtOAc. 0.25 ml (2.6 mmol) diethanol amine was added, and the reactionwas stirred at room temperature overnight. The white ppt which formedwas filtered and sonicated in 1 N HCl. The resulting white ppt wasfiltered, dissolved in MeOH, and concentrated in vacuo. Recovered 36.6mg (13%). HPLC R_(f)=13.912, 1H NMR (DMSO-d₆): 3.85 (s, 3H), 7.15 s,(1H), 7.36 (d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.87 (s, 1H), 8.14(s, 1H), 11.91 (s, 1H).

(5-hydroxyindol-2-yl)-N-[(3-methoxyphenyl)methyl]carboxyamide (5)

Dissolved 2.00 g (11.3 mmol) 5-hydroxy-2-indolecarboxylic acid, 1.6 ml(12.4 mmol) 3 methoxybenzylamine, and 5.87 g (11.3 mmol) PyBOP in 10 mlanh. DMF. Cooled to 0° C. and added 5.9 ml (33.9 mmol) DIEA. Stirred for5 min at 0° C. and allowed to warm to room temperature for 1 hour.Recovered 2.83 g (85% yield) TLC R_(f)=0.34 (1/1 EtOAc/hexanes) 1H NMR(DMSO-d₆): 3.70 (s, 3H), 4.43 (d, J=4.4 Hz, 2H) 6.69 (d, J=8.8 Hz, 1H),6.78 (d, J=7.7 Hz, 1H), 6.83 (s, 1H), 6.86 (s, 1H), 6.94 (s, 1H), 7.20(m, 3H), 8.92 (t, J=4.4 Hz, 1H), 11.36 (s, 1H) FAB(+) MS m/e 297.3 (M+1)

(5-hydroxyindol-2-yl)-N-[(3-hydroxyphenyl)methyl]carboxyamide (6)

Added 20 ml anh. DCM to 200 mg (0.67 mmol)(5-hydroxyindol-2-yl)-N-[(3-methoxyphenyl)methyl]carboxyamide(5) andcooled to −78° C. under argon. Added 4.0 ml (4.0 mmol, 6 eq) BBr₃. Heldat −78° C. for 5 min and warmed to rt. After 90 min at rt, quenched withH₂O and stirred for 10 min. Diluted rxn mix with EtOAc and washed withNaHCO₃ and brine. Dried organic layer over MgSO₄ and concentrated invacuo. Ran through silica plug to remove baseline contamination.Recovered X mg. (80% yield.) TLC R_(f)=0.21 (1/1 EtOAc/hexanes). ¹H NMR(DMSO-d₆): 4.38 (d, J=4.8 Hz, 2H), 6.59 (d, J=8.8 Hz, 1H), 6.71 (m, 3H)6.83 (d, J=1.8 Hz, 1H), 6.94 (s, 1H), 7.08 (dd, J=7.7 Hz, 1H), 7.19 (d,J=8.8 Hz, 1H), 8.84 (t, J=5.9 Hz), 11.28, (s, 1H). FAB(+) MS m/e 283.2(M+1)

N-(1-carbamoyl-2-methylbutyl)(5-hydroxyindol-2-yl)carboxyamide (7)

100 mg (0.56 mmol) 5-hydroxy-2-indolecarboxylic acid, 103.4 mg (0.62mmol, 1.1 eq) L-isoleucinamide, and 291 mg (0.56 mmol, 1 eq) PyBOP wereall dissolved in 1 ml anh DMF. The solution was cooled to 0° C. and 0.3ml (1.68 mmol, 3 eq) DIEA was added. The reaction mixture was stirredfor 1 min at 0° C. and at room temperature for 1 hour. The reaction wasthen diluted with EtOAc and washed with 1 N HCl x3 and sat'd NaHCO3 x 3.The organic layer was dried over MgSO4, and concentrated in vacuo togive 166.7 mg (91% yield.) TLC R_(f)=0.08 (1/1 EtOAc/hexanes). ¹H NMR(DMSO-d₆): 0.83 (m, 6H), 1.15 (m, 2H), 1.68 (m, 1H), 1.83 (m, 1H), 4.29(t, J=8.8 Hz, 1H), 6.69 (d, J=8.5 Hz, 1H), 6.83 (s, 1H), 7.01, (s, 1H),7.06 (s, 1H), 7.19 (d, J=8.4 Hz, 1H), 7.48, (s, 1H), 8.00 (d, 9.2 Hz,1H), 8.76 (s, 1H), 11.3, (s, 1H). FAB(+) MS m/e 290.1 (M+1)

Methyl 5-dibenzylphosphorylindole-2-carboxylate (8)

200 mg (0.62 mmol) methyl5-[(trifluoromethyl)sulfonyloxy]indole-2-carboxylate (2), 195.8 mg (0.74mmol, 1.2 eq) dibenzylphosphite, 0.14 ml (0.81 mmol, 1.3 eq) DIEA, and35.7 mg (0.03 mmol, 5 mol %) Pd(PPh₃)₄ were all dissolved in anh AcCNunder argon. The reaction mix was heated to 80° C. overnight. Thesolvent was removed under reduced pressure, and the title compound wasisolated by silica gel chromatography. 130 mg (50% yield). TLCR_(f)=0.28 (1/1 EtOAc/hexanes) ¹H NMR (DMSO-d₆): 3.85 (s, 3H), 4.98–5.01(m, 4H), 7.28–7.32 (m, 11H), 7.53–7.55 (m, 2H), 8.17 (d, J=14.6 Hz, 1H)³¹P NMR (DMSO-d₆): 23.89.

Methyl 5-phosphonolindole-2-carboxylate

Methyl 5-dibenzylphosphorylindole-2-carboxylate (8) (125 mg) wasdissolved in 10 ml MeOH. 20 mg Pd-C was added and the mixture washydrogenated in a Parr apparatus overnight. Filtered off catalyst andremoved solvent under reduced pressure. Obtained 72.5 mg (73% yield).TLC R_(f) =baseline in EtOAc. ¹H NMR (DMSO-d₆): 3.84 (s, 3H), 7.24 (s,1H), 7.44–7.49 (m, 2H), 8.01 (d, J=14.3 Hz, 1H) 12.11 (s, 1H) ³¹P NMR(DMSO-d₆): 17.22.

The ester compounds in this example could be increased in potency byconverting the ester to an amide and/or adding additional specificityelements.

Example 4 Synthesis of Further Indole Derivative Protein KinaseInhibitors

The synthesis of some further elaborated indole inhibitors isillustrated in below. These syntheses should result in compounds withgreater potency against pp60c-src and other tyrosine kinases. The methylester group can be subsequently converted into various amide derivativesto increase potency.

Example 5 Toxicity of Src inhibitors

There is considerable recent literature support for targetingpp60^(c-src) (Src) as a broadly useful approach to cancer therapywithout resulting in serious toxicity. For example, tumors that displayenhanced EGF receptor PTK signaling, or overexpress the relatedHer-2/neu receptor, have constitutively activated Src and enhanced tumorinvasiveness. Inhibition of Src in these cells induces growth arrest,triggers apoptosis, and reverses the transformed phenotype (Karni etal., 1999). It is known that abnormally elevated Src activity allowstransformed cells to grow in an anchorage-independent fashion. This isapparently caused by the fact that extracellular matrix signalingelevates Src activity in the FAK/Src pathway, in a coordinated fashionwith mitogenic signaling, and thereby blocks an apoptotic mechanismwhich would normally have been activated. Consequently FAK/Srcinhibition in tumor cells may induce apoptosis because the apoptoticmechanism which would have normally become activated upon breaking freefrom the extracellular matrix would be induced (Hisano et al., 1997).Additionally, reduced VEGF mRNA expression was noted upon Src inhibitionand tumors derived from these Src-inhibited cell lines showed reducedangiogenic development (Ellis et al., 1998).

The issue of potential toxicity of Src inhibition has been addressedwith very promising results. For example, a knock-out of the Src gene inmice led to only one defect, namely osteoclasts that fail to formruffled borders and consequently do not resorb bone. However, theosteoclast bone resorb function was rescued in these mice by inserting akinase defective Src gene (Schwartzberg et al., 1997). This suggestedthat Src kinase activity can be inhibited in vivo without triggering theonly known toxicity because the presence of the Src protein isapparently sufficient to recruit and activate other PTKs (which areessential for maintaining osteoclast function) in an osteoclastessential signaling complex.

Src has been proposed to be a “universal” target for cancer therapysince it has been found to be overactivated in a growing number of humantumors, in addition those noted above (Levitzki, 1996). The potentialbenefits of Src inhibition for cancer therapy appear to be four-foldbased upon the cited, and additional, literature. They are: 1)Inhibition of uncontrolled cell growth caused by autocrine growth factorloop effects, etc. 2) Inhibition of metastasis due to triggeringapoptosis upon breaking free from the cell matrix. 3) Inhibition oftumor angiogenesis via reduced VEGF levels. 4) Low toxicity.

The initial non-peptide Src inhibitors have also shown very encouragingresults in four different series of cell culture assays. 1) In the NIH60-tumor cell panel assay, broad activity (as one would expect for a Srcinhibitor) was seen against the tumor cell lines, including the prostatelines. For example, three of the inhibitors gave the following growthinhibition IC₅₀'s against the NIH prostate cancer cell lines: TOM 2-32(PC-3, 15 μM; DU-145,38 μM), TOM 2-47 (PC-3,19 μM), KLM 2-31 (PC-3, 39μM; DU-145, >100 μM). 2) In the v-Src transformed normal rat kidney cellline (LA25) TOM 2-47 & TOM 2-32 specifically blocked the v-Src inducedcell growth without inhibiting the normal growth of the parentnon-transformed cells. This result showed that the inhibitors do notaffect normal cells but are effective in blocking Src induced celltransformation. 3) The Src inhibitors to the cancer drugs etoposide,taxol, doxorubicin and cisplatin in ovarian tumors from three differentpatients and an abdominal carcinoma from another patient. In all cases,the Src inhibitors were at least as effective, and typically moreeffective, than the known cancer drugs, with full efficacy seen at thelowest dose tested (3 μM). As a representative example, a comparison oftaxol and doxorubicin (they were more effective than etoposide &cisplatin in this particular tumor cell culture) with the three Srcinhibitors mentioned above utilizing ovarian tumor cells from tumor N015is shown in FIG. 15A. 4) The Src inhibitors were also tested forinhibition of normal human fibroblast cell growth and found noinhibition of normal cell growth (both subconfluent and confluent; someenhanced growth was observed instead), indicating that these inhibitorsare not toxic to normal cells even at a 10-fold higher concentration. Anexample of his data is given in FIG. 15B.

Overall, the cell data obtained thus far shows what one might expect forSrc inhibitors, i.e. broad activity against many cancer cell lines withlittle or no normal cell toxicity.

The preliminary Src inhibitors are lead structures from which it ispossible to design more potent and selective inhibitors. In addition toutilizing the tyrosine kinase crystal structures, molecular modelingstudies can be carried out with the natural product tyrosine kinaseinhibitor damnacanthal (Faltynek et al., 1995) to investigate itspeptide-competitive binding mode. These additional modeling studies areenable one to design further analogs of Src inhibitors wherein the keypharmacophore elements of damnacanthal are incorporated into the newinhibitors. Their syntheses will be undertaken and the isolated Srctesting done as reported (Marsilje 2000).

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

LITERATURE CITED

The following references which were cited herein, are herebyincorporated by reference into this application:

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1. A method for identifying an inhibitor of a protein kinase comprising:a) providing a first reagent consisting of a peptide scaffold, whereinsaid peptide scaffold is a substrate for the protein kinase, covalentlylinked to a first module comprising one or more functional groupsselected from the group consisting of boronic acid, a hydroxyl group,phosphonic acid, sulfamic acid, a guanidino group, a carboxylic acid, analdehyde, an amide, and hydroxymethylphosphonic acid; b) bringing intocontact the first reagent, a protein kinase, and a natural substrate forthe kinase under conditions sufficient to allow phosphorylation of thesubstrate; c) determining the level of phosphorylation of the substratein the presence of the first reagent and comparing said level to thelevel of phosphorylation in the absence of the first reagent; d)selecting the first reagent that has a lower level of substratephosphorylation in the presence of the first reagent relative to thelevel of phosphorylation of the substrate in the absence of the firstreagent; e) providing a second reagent consisting of a second modulecomprising a non-protein scaffold functional group selected from thegroup consisting of indole, naphthalene, biphenyl, isoquinoline,benzofuran, and benzothiophene, covalently linked to the first module ofthe first reagent selected in step d); f) bringing into contact thesecond reagent, a protein kinase, and a natural substrate for the kinaseunder conditions sufficient to allow phosphorylation of the substrate;g) determining the level of phosphorylation of the substrate in thepresence of the second reagent and comparing said level to the level ofphosphorylation in the absence of the second reagent; h) selecting thesecond reagent that has a lower level of substrate phosphorylation inthe presence of the second reagent relative to the level ofphosphorylation of the substrate in the absence of the second reagent,thereby identifying a protein kinase inhibitor.
 2. The method accordingto claim 1, wherein the first module comprises a boronic acid group. 3.The method according to claim 1, wherein the first module comprises ahydroxyl group.
 4. The method according to claim 1, wherein the firstmodule comprises an amide group.
 5. The method according to claim 4,wherein the amide group is a vicinal tricarbonyl amide.
 6. The methodaccording to claim 1, wherein the first module further comprises alinear chain comprising between one and three carbon atoms which linksthe first module to the second module.
 7. The method according to claim6, wherein one of the carbon atoms in the linear chain is substitutedwith a nitrogen, oxygen or sulfur atom.
 8. The method according to claim1, wherein the second module comprises an indole.
 9. The methodaccording to claim 1, wherein the second module comprises naphthalene.10. The method of claim 1, wherein the peptide scaffold is apentapeptide.
 11. The method of claim 10, wherein the pentapeptide isAc-Ile-Tyr-Gly-Glu-Phe-NH₂ or SEQ ID NO:
 1. 12. The method of claim 1,wherein the pentapeptide is Ac-Ile-Tyr-Gly-Glu-Phe-NH₂ and the firstmodule replaces the tyrosine hydroxyl group.
 13. The method according toclaim 1, wherein the protein kinase is a protein tyrosine kinase. 14.The method according to claim 13, wherein the protein tyrosine kinase isselected from the group consisting of pp60^(c-src), p56^(lck), ZAPkinase, platelet derived growth factor receptor tyrosine kinase,Bcr-Abl, VEGF receptor tyrosine kinase, and epidermal growth factorreceptor tyrosine kinase, and epidermal growth factor receptor-liketyrosine kinases.
 15. The method according to claim 14, wherein theprotein tyrosine kinase is pp60^(c-src).
 16. The method according toclaim 1, wherein the protein kinase is a protein serine kinase.
 17. Themethod according to claim 16, wherein the protein serine kinase isselected from the group consisting of MAP kinase, protein kinase C, andCDK kinase.
 18. The method according to claim 1, further comprising: i)providing a third reagent consisting of the second reagent selected instep h) covalently linked to one or more specificity elements (S)_(n);j) bringing into contact the third reagent, a protein kinase, and anatural substrate for the kinase under conditions sufficient to allowphosphorylation of the substrate; k) determining the level ofphosphorylation of the substrate in the presence of the third reagentand comparing said level to the level of phosphorylation in the absenceof the third reagent; l) selecting the third reagent that has a lowerlevel of substrate phosphorylation in the presence of the third reagentrelative to the level of phosphorylation of the substrate in the absenceof the third reagent, thereby identifying a protein kinase inhibitor.19. The method of claim 1, wherein the first reagent in step a) isrationally designed using molecular modeling.
 20. The method of claim 1,wherein the second reagent in step e) is rationally designed usingmolecular modeling.
 21. The method of claim 18, wherein the thirdreagent in step i) is rationally designed using molecular modeling. 22.The method of claim 18 wherein the one or more specificity elements areselected from an amine, an alkyl group, a hydroxyl group, an amide, anester, and 3-aminophenol.
 23. The method of claim 1, wherein theconditions sufficient to allow phosphorylation are Literature Mimeticconditions.
 24. The method of claim 1, wherein the conditions sufficientto allow phosphorylation are Cellular Mimetic conditions.